Genetic modification of rats

ABSTRACT

Compositions and methods are provided for making rat pluripotent and totipotent cells, including rat embryonic stem (ES) cells. Compositions and methods for improving efficiency or frequency of germline transmission of genetic modifications in rats are provided. Such methods and compositions comprise an in vitro culture comprising a feeder cell layer and a population of rat ES cells or a rat ES cell line, wherein the in vitro culture conditions maintain pluripotency of the ES cell and comprises a media having mouse leukemia inhibitory factor (LIF) or an active variant or fragment thereof. Various methods of establishing such rat ES cell lines are further provided. Methods of selecting genetically modified rat ES cells are also provided, along with various methods to generate a transgenic rat from the genetically modified rat ES cells provided herein. Various kits and articles of manufacture are further provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/242,025, filed Aug. 19, 2016, which is a divisional application ofU.S. patent application Ser. No. 14/185,703, filed Feb. 20, 2014, whichclaims priority to U.S. Provisional Application No. 61/767,093, filedFeb. 20, 2013, each of which is herein incorporated by reference in itsentirety for all purposes.

FIELD

Non-human pluripotent, totipotent, and embryonic stem (ES) cells, inparticular rat pluripotent, totipotent, and/or rat ES cells, and methodsof making them. Methods for making rat pluripotent, totipotent, and EScells are provided. Methods for targeting rat pluripotent, totipotent,and/or ES cells are provided. Methods for achieving germlinetransmission of a genetic modification in a rat cell are provided. Mediafor deriving, growing, and maintaining rat pluripotent, totipotent, andES cells are provided.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted concurrently withthe specification as a text file via EFS-Web, in compliance with theAmerican Standard Code for Information Interchange (ASCII), with a filename of 529169SEQLIST.txt, a creation date of May 1, 2019, and a size of2.11 kb. The sequence listing filed via EFS-Web is part of thespecification and is hereby incorporated in its entirety by referenceherein.

BACKGROUND

The rat has been a valuable model for many applications, including, butnot limited to, applications in drug discovery. The usefulness of therat has been mitigated somewhat by difficulty in obtaining geneticallymodified rats, in particular, in developing methods for geneticallymodifying rats, and generating useful rat cells that can be used ingenetic modification protocols, including but not limited to protocolsthat result in germline transmission of a genetic modification in a ratgenome.

There is a need in the art for rat cells (e.g., embryonic stem cells)that can be genetically modified such that the genetic modification canbe transmitted through the germline. There is a need in the art forimproved frequency of germline transmission of genetic modifications inrats.

There is a need in the art for donor rat pluripotent, totipotent, and/orES cells from various strains of rat that are capable of generating F0,or wholly donor cell-derived, F0 rats. There is a need in the art fordonor rat pluripotent, totipotent, and/or ES cells that are capable ofgenerating rats that comprise a germline genetic modification.

SUMMARY

Compositions and methods are provided for making rat pluripotent and/ortotipotent cells, including rat embryonic stem (ES) cells. Compositionsand methods for improving efficiency or frequency of germlinetransmission of genetic modifications in rats are provided. In variousaspects, the methods and compositions comprise an in vitro culturecomprising a feeder cell layer and a population of rat ES cells or a ratES cell line, wherein the in vitro culture conditions allow maintenanceof pluripotency of the rat ES cell. Various methods of establishing ratES cell lines are further provided. Method of selecting geneticallymodified rat ES cells are also provided, along with various methods togenerate a transgenic rat from the genetically modified rat ES cells areprovided herein. Various kits and articles of manufacture are furtherprovided.

Non-limiting embodiments are as follows:

1. An isolated rat ES cell of a strain selected from ACI or DA, whereinthe isolated rat ES cell is and capable of transmitting its genomethrough the germline.

2. The isolated rat ES cell of embodiment 1, wherein the cell is derivedfrom an ACI rat.

3. The isolated rat ES cell of embodiment 1 or 2, wherein the cell isderived from a Dark Agouti (DA) rat.2.

4. The isolated rat ES cell of embodiment 1, 2, or 3, wherein the cellis euploid and capable of transmitting a targeted genetic modificationthrough the germline.

5. The isolated rat ES cell of embodiment 4, wherein the rat ES cellcomprises a germline transmission efficiency of the targeted geneticmodification of at least 3%.

6. The isolated rat ES cell of embodiment 4, wherein the rat ES cell hasa germline transmission efficiency of the targeted genetic modificationof at least 60%.

7. The isolated rat ES cell of any one of embodiments 1-6, wherein therat ES cell exhibits a targeting efficiency of homologous recombinationof at least 2%.

8. The isolated rat ES cell of any one of embodiments 1-8, wherein therat ES cell is capable of transmitting a targeted genetic modificationinto progeny following a successive round of electroporation.

9. The isolated rat ES cells of any one of embodiments 1-8, wherein therat ES cell comprises one or more, two or more, or three or moretargeted genetic modification.

10. The isolated rat ES cell of any one of embodiments 4-9, wherein thetargeted genetic modification comprises an insertion, a deletion, aknockout, a knockin, a point mutation, or a combination thereof.

11. The isolated rat ES cell of embodiment 9, wherein the targetedgenetic modification comprises at least one insertion of a heterologouspolynucleotide into a genome of the cell.

12. The isolated rat ES cell of embodiment 11, wherein the heterologouspolynucleotide comprises a selection marker.

13. The isolated rat ES cell of embodiment 12, wherein (a) the selectionmarker comprises a non-attenuated selection marker gene operably linkedto a promoter; or (b) the rat ES cell comprises at least 2 copies of thepolynucleotide encoding the selection marker.

14. The isolated rat ES cell of embodiment 12, wherein the selectionmarker has an increased activity compared to a wild type selectionmarker.

15. The isolated rat ES cell of any one of embodiments 1-14, wherein therat ES cell forms a sphere-like colony when plated on a feeder celllayer in culture comprising a LIF polypeptide, a GSK3 inhibitor, and aMEK inhibitor.

16. The isolated rat ES cell of any one of embodiments 1-15, wherein therat ES cell, when cultured in vitro, loosely adhere to the feeder celllayer.

17. The isolated rat ES cell of any one of embodiments 1-16, wherein thecell does not require paracrine LIF signaling for maintenance ofpluripotency.

18. The isolated rat ES cell of any one of embodiments 1-17, wherein thecell is a male (XY) rat ES cell.

19. The isolated rat ES cell of any one of embodiments 1-19, wherein thecell is a female (XX) rat ES cell.

20. The isolated rat ES cell of any one of embodiments 1-19, wherein therat ES cell can be passaged up to at least 11 times in a mediumcomprising a GSK3 inhibitor and a MEK inhibitor without decreasing itstargeting efficiency or germline transmission efficiency of a targetedgenetic modification.

21. The isolated rat ES cell of any one of embodiments 1-20, wherein therat ES cells express at least one pluripotency marker selected fromDnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIF receptor,Lin28, Nanog, Oct4, Sox15, Sox2, Utf1, or a combination thereof.

22. The isolated rat ES cell of any one of embodiments 1-21, wherein therat ES cells do not express one or more pluripotency markers selectedfrom c-Myc, Ecat1, Rexo1, or a combination thereof.

23. The isolated rat ES cell of any one of embodiments 1-22, wherein therat ES cells do not express one or more mesodermal markers selected fromBrachyury, Bmpr2, or a combination thereof.

24. The isolated rat ES cell of any one of embodiments 1-23, wherein therat ES cells do not express one or more endodermal markers selected fromGata6, Sox17, Sox7, or combination thereof;

25. The isolated rat ES cell of any one of embodiments 1-24, wherein therat ES cells do not express one or more neural markers selected fromNestin, Pax6, or combination thereof.

26. The isolated rat ES cell of any one of embodiments 1-25, wherein thecell expresses a pluripotency marker comprising Oct-4, Sox2, alkalinephosphatase, or a combination thereof.

27. The isolated rat ES cell of any one of embodiments 1-26, wherein therat ES cell is characterized by the expression of one or more of a ratESC-specific gene selected from one or more of Adherens JunctionsAssociate Protein 1 (Ajap1), Claudin 5 (Cldn5), Cdc42 guanine nucleotideexchange factor 9 (Arhgef9), Calcium/calmodulin-dependent protein kinaseIV (Camk4), ephrin-A1 (Efna1), EPH receptor A4 (Epha4), gap junctionprotein beta 5 (Gjb5), Insulin-like growth factor binding protein-like 1(Igfbpl1), Interleukin 36 beta (Il1f4 Interleukin 28 receptor, alpha(Il28ra), left-right determination factor 1 (Lefty1), Leukemiainhibitory factor receptor alpha (Lifr), Lysophosphatidic acid receptor2 (Lpar2), Neuronal pentraxin receptor (Ntm), Protein tyrosinephosphatase non-receptor type 18 (Ptpn18), Caudal type homeobox 2(Cdx2), Fibronectin type III and ankyrin repeat domains 1 (Fank1),Forkhead box E1 (thyroid transcription factor 2) (Foxe1),Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Forkhead boxE1 (thyroid transcription factor 2) (Foxe1), Hairy/enhancer-of-splitrelated with YRPW motif 2 (Hey2), Lymphoid enhancer-binding factor 1(Lef1), Sal-like 3 (Drosophila) (Sall3), SATB homeobox 1 (Satb1),miR-632, or a combination thereof.

28. An isolated population of rat ES cells, wherein at least 70% of therat ES cells are euploid and form sphere-like colonies when plated on afeeder cell layer in vitro.

29. The isolated population of rat ES cells of embodiment 28, whereinthe rat ES cells are derived from an ACI rat.

30. The isolated population of rat ES cells of embodiment 28, whereinthe rat ES cells are derived from a Dark Agouti (DA) rat.

31. The isolated population of rat ES cells of any one of embodiments28-30, wherein the rat ES cells are capable of transmitting their genomethrough the germline.

32. The isolated population of rat ES cells of any one of embodiments28-31, wherein the rat ES cells have a germline transmission efficiencyof the targeted genetic modification of at least 3%.

33. The isolated population of rat ES cells of any one of embodiments28-31, wherein the rat ES cells have a germline transmission efficiencyof the targeted genetic modification of at least 60%.

34. The isolated population of rat ES cells of any one of embodiments28-31, wherein the rat ES cells exhibit a targeting efficiency ofhomologous recombination of at least 2%.

35. The isolated population of rat ES cells of any one of embodiments28-34, wherein the rat ES cells are capable of transmitting a targetedgenetic modification into progeny following a successive round ofelectroporation.

36. The isolated population of any one embodiments 28-35, wherein therat ES cells comprise one or more, two or more, or three or moretargeted genetic modification and can transmit the targeted geneticmodification through the germline.

37. The isolated population of rat ES cells of embodiment 36, whereinthe targeted genetic modification is at the rat Rosa26 locus.

38. The isolated population of rat ES cells of embodiment 36, whereinthe targeted genetic modification comprises an insertion, a deletion, aknockout, a knockin, a point mutation, or a combination thereof.

39. The isolated population of rat ES cells of embodiment 36, whereinthe targeted genetic modification comprises at least one insertion of aheterologous polynucleotide into a genome of the cell.

40. The isolated population of rat ES cells of embodiment 39, whereinthe heterologous polynucleotide comprises a selection marker.

41. The isolated population of rat ES cells of embodiment 40, wherein

(a) the selection marker comprises a non-attenuated selection markergene operably linked to a promoter; or

(b) the rat ES cell comprises at least 2 copies of the polynucleotideencoding the selection marker.

42. The isolated population of rat ES cells of embodiment 40, whereinthe selection marker has an increased activity compared to a wild typeselection marker.

43. The isolated population of rat ES cells of any one of embodiments28-42, wherein the cells form a sphere-like colony when plated on afeeder cell layer in culture comprising a LIF polypeptide, a GSK3inhibitor, and a MEK inhibitor.

44. The isolated population rat ES cells of any one of embodiments28-43, wherein the cells, when cultured in vitro, loosely adhere to thefeeder cell layer.

45. The isolated population of rat ES cells of any one of embodiments28-44, wherein the cells do not require paracrine LIF signaling formaintenance of pluripotency.

46. The isolated population of rat ES cells of any one of embodiments28-44, wherein the rat ES cells are a male (XY) rat ES cells.

47. The isolated population of rat ES cells of any one of embodiments28-44, wherein the rat ES cells are female (XX) rat ES cells.

48. The isolated population of rat ES cells of any one of embodiments28-47, wherein the rat ES cells can be passaged up to at least 11 timesin a medium comprising a GSK3 inhibitor and a MEK inhibitor withoutdecreasing its targeting efficiency or germline transmission efficiencyof a targeted genetic modification.

49. The isolated population of rat ES cells of any one of embodiments28-48, wherein the rat ES cells express at least one pluripotency markerselected from Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1,LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, Utf1, or a combinationthereof.

50. The isolated population of rat ES cells of any one of embodiments28-49, wherein the rat ES cells do not express one or more pluripotencymarkers selected from c-Myc, Ecat1, Rexo1, or a combination thereof.

51. The isolated population of rat ES cells of any one of embodiments28-50, wherein the rat ES cells do not express one or more mesodermalmarkers selected from Brachyury, Bmpr2, or a combination thereof.

52. The isolated population of rat ES cells of any one of embodiments28-51, wherein the rat ES cells do not express one or more endodermalmarkers selected from Gata6, Sox17, Sox7, or combination thereof;

53. The isolated population of rat ES cells of any one of embodiments28-52, wherein the rat ES cells do not express one or more neuralmarkers selected from Nestin, Pax6, or combination thereof.

54. The isolated population of rat ES cells of any one of embodiment28-53, wherein the rat ES cells expresses a pluripotency markercomprising Oct-4, Sox2, alkaline phosphatase, or a combination thereof.

55. The isolated population of rat ES cells of any one of embodiments28-54, wherein the rat ES cells are characterized by the expression ofone or more of a rat ESC-specific gene selected from one or more ofAdherens Junctions Associate Protein 1 (Ajap1), Claudin 5 (Cldn5), Cdc42guanine nucleotide exchange factor 9 (Arhgef9),Calcium/calmodulin-dependent protein kinase IV (Camk4), ephrin-A1(Efna1), EPH receptor A4 (Epha4), gap junction protein beta 5 (Gjb5),Insulin-like growth factor binding protein-like 1 (Igfbpl1), Interleukin36 beta (Il1f8), Interleukin 28 receptor, alpha (Il28ra), left-rightdetermination factor 1 (Lefty1), Leukemia inhibitory factor receptoralpha (Lifr), Lysophosphatidic acid receptor 2 (Lpar2), Neuronalpentraxin receptor (Ntm), Protein tyrosine phosphatase non-receptor type18 (Ptpn18), Caudal type homeobox 2 (Cdx2), Fibronectin type III andankyrin repeat domains 1 (Fank1), Forkhead box E1 (thyroid transcriptionfactor 2) (Foxe1), Hairy/enhancer-of-split related with YRPW motif 2(Hey2), Forkhead box E1 (thyroid transcription factor 2) (Foxe1),Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Lymphoidenhancer-binding factor 1 (Lef1), Sal-like 3 (Drosophila) (Sall3), SATBhomeobox 1 (Satb1), miR-632, or a combination thereof.

56. The isolated population of rat ES cells of any one of embodiments28-55, wherein the population comprises at least 10⁴ cells.

57. The isolated population of rat ES cells of any one of embodiments28-56, wherein the rat ES cells have one or more characteristiccomprising:

a. at least 90% of the rat ES cells are euploid;

b. at least 70% of the rat ES cells express at least one pluripotencymarker; wherein the at least one pluripotency marker comprises Oct-4,Sox2, alkaline phosphatase, or a combination thereof;

c. a cell from the rat ES cell population, when combined with a rat hostembryo transmits the genome of the rat ES cell line into an offspring;

d. the rat ES cells when cultured in vitro loosely adhere to a feedercell layer;

e. the rat ES cells form sphere-like colonies when plated on a feedercell layer in vitro; (f) the rat ES cells maintain pluripotency whencultured in vitro in a media comprising an GSK3 inhibitor, a MEKinhibitor, LIF and a feeder cell layer that is not genetically modifiedto express LIF;

f. the rat ES cell exhibits a targeting efficiency of homologousrecombination of at least 2%;

g. the rat ES cells maintain pluripotency in vitro without requiringparacrine LIF signaling;

h. at least 70% of the rat ES cells are euploid and form sphere-likecolonies when plated on a feeder cell layer in vitro;

i. the rat ES cells express at least one pluripotency marker selectedfrom Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIFreceptor, Lin28, Nanog, Oct4, Sox15, Sox2, Utf1, or a combinationthereof;

j. the rat ES cells do not express one or more differentiation markersselected from c-Myc, Ecat1, Rexo1;

k. the rat ES cells do not express one or more mesodermal markersselected from Brachyury, Bmpr2, or a combination thereof;

1. the rat ES cells do not express one or more endodermal markersselected from Gata6, Sox17, Sox7, or combination thereof; and/or

m. the rat ES cells do not express one or more neural markers selectedfrom Nestin, Pax6, or combination thereof.

58. The isolated population of rat ES cells of any one of embodiments28-57, wherein (a) the rat ES cells is derived from a rat blastocyst;(b) the rat ES cell is derived from a rat morula stage embryo; and/or,(c) the rat ES cell line is derived from a superovulated rat.

59. An in vitro culture comprising a feeder cell layer, the populationof rat embryonic stem (ES) cells, and a medium comprising a LeukemiaInhibitory Factor (LIF), GSK3 inhibitor, and a MEK inhibitor, wherein atleast 70% of the rat ES cells are euploid and the rat ES cell forms asphere-like colony.

60. The in vitro culture of embodiment 59 or 60, wherein the rat EScell, loosely adhere to the feeder cell layer.

61. The in vitro culture of embodiment 59, 60, or 61, wherein the rat EScells are capable of transmitting their genome through the germline.

62. The in vitro culture of embodiment 59, 60 or 61, wherein the rat EScells are derived from an ACI rat.

63. The in vitro culture of embodiment 59, 60 or 61, wherein the rat EScells are derived from a Dark Agouti (DA) rat.2.

64. The in vitro culture of any one of embodiments 59-63, wherein therat ES cells are capable of transmitting a targeted genetic modificationthrough the germline.

65. The in vitro culture of embodiment 64, wherein the rat ES cellscomprise a germline transmission efficiency of the targeted geneticmodification of at least 3%.

66. The in vitro culture of embodiment 64, wherein the rat ES cells havea germline transmission efficiency of the targeted genetic modificationof at least 60%.

67. The in vitro culture of any one of embodiments 59-66, wherein therat ES cells exhibit a targeting efficiency of homologous recombinationof at least 2%.

68. The in vitro culture of any one of embodiments 59-67, wherein therat ES cell is capable of transmitting a targeted genetic modificationinto progeny following a successive round of electroporation.

69. The in vitro culture of any one of embodiments 59-68, wherein therat ES cell comprises one or more, two or more, or three or moretargeted genetic modification.

70. The in vitro culture of embodiment 69, wherein the targeted geneticmodification comprises an insertion, a deletion, a knockout, a knockin,a point mutation, or a combination thereof.

71. The in vitro culture of embodiment 69, wherein the targeted geneticmodification comprises at least one insertion of a heterologouspolynucleotide into a genome of the cell.

72. The in vitro culture of embodiment 71, wherein the heterologouspolynucleotide comprises a selection marker.

73. The in vitro culture of embodiment 72, wherein (a) the selectionmarker comprises a non-attenuated selection marker gene operably linkedto a promoter; or (b) the rat ES cell comprises at least 2 copies of thepolynucleotide encoding the selection marker.

74. The in vitro culture of embodiment 72, wherein the selection markerhas an increased activity compared to a wild type selection marker.

75. The in vitro culture of any one of embodiments 59-74, wherein thecell does not require paracrine LIF signaling for maintenance ofpluripotency.

76. The in vitro culture of embodiment of any one of embodiments 59-75,wherein the cell is a male (XY) rat ES cell.

77. The in vitro culture of embodiment of any one of embodiments 59-75,wherein the cell is a female (XX) rat ES cell.

78. The in vitro culture of embodiment of any one of embodiments 59-77,wherein the rat ES cell can be passaged up to at least 11 times in amedium comprising a GSK3 inhibitor and a MEK inhibitor withoutdecreasing its targeting efficiency or germline transmission efficiencyof a targeted genetic modification.

79. The in vitro culture of any one of embodiments 59-78, wherein therat ES cells express at least one pluripotency marker selected fromDnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIF receptor,Lin28, Nanog, Oct4, Sox15, Sox2, Utf1, or a combination thereof.

80. The in vitro culture of any one of embodiments 59-79, wherein therat ES cells do not express one or more pluripotency markers selectedfrom c-Myc, Ecat1, Rexo1, or a combination thereof.

81. The in vitro culture of any one of embodiments 59-80, wherein therat ES cells do not express one or more mesodermal markers selected fromBrachyury, Bmpr2, or a combination thereof.

82. The in vitro culture of any one of embodiments 59-81, wherein therat ES cells do not express one or more endodermal markers selected fromGata6, Sox17, Sox7, or combination thereof;

83. The in vitro culture of any one of embodiments 59-82, wherein therat ES cells do not express one or more neural markers selected fromNestin, Pax6, or combination thereof.

84. The in vitro culture of any one of embodiments 59-83, wherein thecell expresses a pluripotency marker comprising Oct-4, Sox2, alkalinephosphatase, or a combination thereof.

85. The in vitro culture of any one of embodiments 59-84, wherein therat ES cells are characterized by the expression of one or more of a ratESC-specific gene selected from one or more of Adherens JunctionsAssociate Protein 1 (Ajap1), Claudin 5 (Cldn5), Cdc42 guanine nucleotideexchange factor 9 (Arhgef9), Calcium/calmodulin-dependent protein kinaseIV (Camk4), ephrin-A1 (Efna1), EPH receptor A4 (Epha4), gap junctionprotein beta 5 (Gjb5), Insulin-like growth factor binding protein-like 1(Igfbpl1), Interleukin 36 beta (Il1f4 Interleukin 28 receptor, alpha(Il28ra), left-right determination factor 1 (Lefty1), Leukemiainhibitory factor receptor alpha (Lifr), Lysophosphatidic acid receptor2 (Lpar2), Neuronal pentraxin receptor (Ntm), Protein tyrosinephosphatase non-receptor type 18 (Ptpn18), Caudal type homeobox 2(Cdx2), Fibronectin type III and ankyrin repeat domains 1 (Fank1),Forkhead box E1 (thyroid transcription factor 2) (Foxe1),Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Forkhead boxE1 (thyroid transcription factor 2) (Foxe1), Hairy/enhancer-of-splitrelated with YRPW motif 2 (Hey2), Lymphoid enhancer-binding factor 1(Lef1), Sal-like 3 (Drosophila) (Sall3), SATB homeobox 1 (Satb1),miR-632, or a combination thereof.

86. The in vitro culture of any one of embodiments 59-84, wherein theconcentration of LIF is 50 U/ml to 150 U/ml.

87. The in vitro culture of any one of embodiments 59-85, wherein theconcentration of LIF is 100 U/ml.

88. The in vitro culture of any one of embodiments 59-87, wherein theLIF is from mouse or comprises at least 92% sequence identity to SEQ IDNO: 1.

89. The in vitro culture of any one of embodiments 59-88, wherein therat ES cell is capable of maintaining a pluripotency without requiring aparacrine LIF signaling.

90. The in vitro culture of any one of embodiments 59-89, wherein thefeeder cell layer is not genetically modified to express LIF.

91. The in vitro culture of any one of embodiments 59-90, wherein thefeeder cell layer comprises a monolayer of mitotically inactivated mouseembryonic fibroblasts (MEFs)

92. The in vitro culture of any one of embodiments 59-91, wherein theMEK inhibitor comprises PD0325901.

93. The in vitro culture of any one of embodiment 59-92, wherein theGSK-3 inhibitor comprises CHIR99021.

94. The in vitro culture of any one of embodiments 59-93, wherein thepopulation of rat ES cells is derived from a rat blastocyst-stage embryoor a rat morula-stage embryo.

95. The in vitro culture of embodiment 94, wherein the blastocyst-stageor the morula-stage rat embryo further comprises an outgrowth of anamorphous undifferentiated mass of rat ES cells.

96. The in vitro culture of embodiment 94, wherein the population of ratES cells comprises an isolated outgrowth of an amorphousundifferentiated mass of rat ES cells.

97. A method for generating a rat embryonic stem (ES) cell linecomprising: (a) culturing in vitro a first feeder cell layer and amorula or a blastocyst-stage rat embryo, wherein the zona pellucida ofthe morula or blastocyst-stage rat embryo has been removed, and whereinthe culture conditions maintain pluripotency of a rat ES cell andcomprise a medium having mouse leukemia inhibitory factor (LIF) or asequence having at least 91% sequence identity to SEQ ID NO:1 and havingLIF activity, and a GSK3 inhibitor, and a MEK inhibitor; and, (b)transferring an outgrowth of an amorphous undifferentiated mass of ratES cells to an in vitro culture well comprising a second feeder celllayer and culturing the outgrowth under conditions comprising the mediumhaving the mouse LIF or an active variant of the mouse LIF, and therebymaintaining pluripotency of the rat ES cells; and, establishing a rat EScell line therefrom.

98. The method of embodiment 97, wherein the rat ES cell line ispassaged at least 5 times.

99. The method of embodiment 97 or 98, wherein the rat ES cell line ispassaged at least 10 times.

100. The method of embodiment 97, 98, or 99, wherein the mediumcomprises about 50 U/ml to about 150 U/ml of mouse LIF.

101. The method of any one of embodiments 97-100, wherein the mediumcomprises about 100 U/ml of mouse LIF.

102. The method of any one of embodiments 97-101, wherein the feedercell layer is not genetically modified to express LIF.

103. The method of any one of embodiments 97-102, wherein the feedercell layer comprises a monolayer of mitotically inactivated mouseembryonic fibroblasts (MEFs).

104. The method of any one of embodiments 97-103, wherein the MEKinhibitor comprises PD0325901.

105. The method of any one of embodiments 97-104, wherein the GSK-3inhibitor comprises CHIR99021.

106. The method of any one of embodiment 97-105, wherein (a) the rat EScell line is derived from an ACI rat or derived from a Dark Agouti (DA)rat; (b) the rat ES cell line is derived from a morula-stage or ablastocyst-stage rat embryo; and/or, (c) the rat ES cell line is derivedfrom a morula-stage or a blastocyst-stage embryo from a superovulatedrat.

107. The method of any one of embodiments 97-106, wherein the mediumfurther comprises at least one of an FGF receptor inhibitor, a ROCKinhibitor, or an ALK inhibitor.

108. The method of embodiment 107, wherein the FGF receptor inhibitorcomprises PD184352, the ROCK inhibitor comprises Y-27632, or the ALKinhibitor comprises A-83-01.

109. The method any one of embodiments 97-108, wherein at least one ratES cell has a germline transmission efficiency of the targeted geneticmodification is at least 3%.

110. The method of embodiment any one of embodiments 97-109, wherein thegermline transmission efficiency of the targeted genetic modification isat least 60%.

111. A method of selecting a rat embryonic stem (ES) cells having stablyincorporated into its genome a heterologous polynucleotide comprising:(a) providing an in vitro population of rat ES cells; (b) introducinginto at least one rat ES cell a heterologous polynucleotide comprising aselection marker operably linked to a promoter active the rat ES cell;and, (c) culturing in vitro the rat ES cell population in an alternatingfirst and second culture media, wherein the first culture mediumcomprises an effective amount of a selection agent for a first timeperiod and the second culture medium does not comprise the selectionagent, wherein the in vitro culture conditions are sufficient tomaintain pluripotency; thereby selecting the rat ES cell having stablyintegrated into its genome the heterologous polynucleotide.

112. The method of embodiment 111, wherein the first and second culturemedia are alternated every 24 hours.

113. The method of embodiment 111 or 112, wherein the selection markerimparts resistance to an antibiotic.

114. The method of any one of embodiments 111-113, wherein theantibiotic comprises G418.

115. The method of any one of embodiment 111-114, wherein the selectionmarker comprises neomycin phosphotransferase (neo^(r)), hygromycin Bphosphotransferase (hyg^(r)), puromycin-N-acetyltransferase (puro^(r)),blasticidin S deaminase (bsr^(r)), xanthine/guanine phosphoribosyltransferase (gpt), and herpes simplex virus thymidine kinase (HSV-k), ora combination thereof.

116. The method of any one of embodiments 111-115, wherein (a) theselection marker has an increased activity compared to the wild typeselection marker; and/or (b) multiple copies of the selection marker arestably incorporated into the genome of the rat ES cell.

117. The method of embodiment 116, wherein the selection marker is anon-attenuated selection marker.

118. A method for genetically modifying an isolated rat embryonic stem(ES) cell comprising introducing into the genome of an isolated rat EScell of any one of embodiment 1-58 a heterologous polynucleotide to forma genetically modified rat ES cell.

119. A method of making a genetically modified rat comprising:

(a) introducing into the genome of the isolated rat embryonic stem (ES)cell of any one of embodiments 1-58, a heterologous polynucleotide toform a rat ES cell having a genetic modification;

(b) introducing at least one of the rat ES cells comprising the targetedgenetic modification into a rat host embryo to produce an F0 embryo;

(c) implanting the F0 embryo into a surrogate mother;

(d) gestating the F0 embryo in the surrogate mother to term; and, (e)identifying an F0 rat having the targeted genetic modification.

120. The method of embodiment 119, further comprising breeding a male F0rat with a wild type female rat to produce an F1 progeny that isheterozygous for the targeted genetic modification.

121. The method of embodiment 120, further comprising breeding a male F0rat with a wild type female rat to produce an F1 progeny that isheterozygous for the targeted genetic modification.

122. The method of embodiment 119, further comprising breeding a malerat of the F1 progeny with a female rat of the F1 progeny to obtain anF2 progeny that is homozygous for the genetic modification.

123. The method of any one of embodiments 119-122, wherein at least 3%of the F0 rats having the genetic modification transmit the geneticmodification to the F1 progeny.

124. The method of any one of embodiments 119-123, wherein at least 10%of the F0 rats having the genetic modification transmit the geneticmodification to the F1 progeny.

125. The method of any one of embodiments 119-124, wherein at least 60%of the F0 rats having the genetic modification transmit the geneticmodification to the F1 progeny.

126. The method of any one of embodiments 119-125, wherein thegenetically modified rat ES cell is from the same rat strain as the rathost embryo.

127. The method of any one of embodiments 119-127, wherein thegenetically modified rat ES cell is from a different rat strain as therat host embryo.

128. The isolated population of rat ES cells of any of the precedingclaims, the in vitro culture of any of the preceding claims, or themethod of any of the preceding claims, wherein the rat ES cells in thepopulation comprise:

(a) at least 90% of the rat ES cells are euploid;

(b) at least 70% of the rat ES cells express at least one pluripotencymarker; wherein the at least one pluripotency marker comprises Oct-4,Sox2, alkaline phosphatase, or a combination thereof;

(c) a cell from the rat ES cell population, when combined with a rathost embryo transmits the genome of the rat ES cell line into anoffspring;

(d) the rat ES cells when cultured in vitro loosely adhere to a feedercell layer;

(e) the rat ES cells form sphere-like colonies when plated on a feedercell layer in vitro;

(f) the rat ES cells maintain pluripotency when cultured in vitro in amedia comprising an GSK3 inhibitor, a MEK inhibitor, LIF and a feedercell layer that is not genetically modified to express LIF;

(g) the rat ES cell exhibits a targeting efficiency of homologousrecombination of at least 2%;

(h) the rat ES cells maintain pluripotency in vitro without requiringparacrine LIF signaling;

(i) at least 70% of the rat ES cells are euploid and form sphere-likecolonies when plated on a feeder cell layer in vitro;

(j) the rat ES cells express at least one pluripotency marker selectedfrom Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIFreceptor, Lin28, Nanog, Oct4, Sox15, Sox2, Utf1, or a combinationthereof;

(k) the rat ES cells do not express one or more differentiation markersselected from c-Myc, Ecat1, Rexo1.

(l) the rat ES cells do not express one or more mesodermal markersselected from Brachyury, Bmpr2, or a combination thereof;

(m) the rat ES cells do not express one or more endodermal markersselected from Gata6, Sox17, Sox7, or combination thereof; and/or

(n) the rat ES cells do not express one or more neural markers selectedfrom Nestin, Pax6, or combination thereof.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 depicts rESCs, which grow as compact spherical colonies thatroutinely detach and float in the dish.

FIG. 2A through D depicts various pluripotency markers expressed byrESCs: FIG. 2A depicts Oct-4 (green); FIG. 2B depicts Sox-2 (red); FIG.2C depicts DAPI (blue); FIG. 2D depicts an overlay of pluripotencymarkers expressed by rESCs.

FIG. 3 depicts that the rESCs express light levels of alkalinephosphatase (a pluripotency marker) (left), and the karyotype for lineDA.2B is 42X,Y (right). Karyotyping was done because rESCs often becometetraploid; lines were thus pre-screened by counting metaphasechromosome spreads, and lines with mostly normal counts were thenformally karyotyped.

FIG. 4 depicts a closer view of a rESC of FIG. 1.

FIG. 5 depicts production of chimeras by blastocyst injection andtransmission of the rESC genome through the germline; chimeras producedby blastocyst injection using parental ACI.G1 rESC; high percentagechimeras usually have albino snouts.

FIG. 6 depicts F1 agouti pups with albino littermates, sired by ACI/SDchimera labeled with an asterisk (*) in FIG. 5.

FIG. 7A depicts targeting of the rat Rosa 26 locus, which lies betweenthe Setd5 and Thumpd3 genes as in mouse, with the same spacing. FIG. 7Ashows the structure of the mouse Rosa 26 locus. mRosa26 transcriptsconsist of 2 or 3 exons. FIG. 7B depicts the structure of the rRosa26locus; the rat locus contains a second exon 1 (Ex1b) in addition to thehomologous exon to mouse exon1 (Ex1a); no third exon has been identifiedin rat. FIG. 7C depicts a targeted rRosa26 allele; homology arms of 5 kbeach were cloned by PCR using genomic DNA from DA rESC; the targetedallele contains a SA-lacZ-hUB-neo cassette replacing a 117 bp deletionin the rRosa26 intron.

FIG. 8A depicts a control brain of a 14-week-old wild type rat, whichwas treated with X-gal. The control brain showed a low level ofbackground staining for LacZ (dorsal view).

FIG. 8B depicts LacZ expression in the brain of an rRosa26 heterozygousrat (14-week old). The lacZ reporter was expressed ubiquitouslythroughout the brain of the rRosa26 heterozygote.

FIG. 8C depicts a control heart and thymus (inset) of a 14-week-old wildtype rat, which were treated with X-gal. The control heart and thymusshowed a low level of background staining for LacZ.

FIG. 8D depicts LacZ expression in the heart and thymus (inset) of a14-week-old rRosa26 heterozygous rat. The lacZ reporter was expressedubiquitously throughout the heart and thymus of the rROSA26heterozygote.

FIG. 8E depicts a control lung of a 14-week-old wild type rat, whichwere treated with X-gal. The control lung showed a low level ofbackground staining for LacZ.

FIG. 8F depicts LacZ expression in the lung of a 14-week-old rRosa26heterozygote rat. The lacZ reporter was expressed ubiquitouslythroughout the lung of the rRosa26 heterozygote.

FIGS. 8G and H depict LacZ expression in e12.5 embryos. In contrast tothe wild-type control embryo (FIG. 8H), which shows a low level ofbackground LacZ staining, the rRosa26 heterozygous embryo exhibitedubiquitous expression of the LacZ reporter throughout the embryo.

FIGS. 8I and J depict LacZ expression in e14.5 embryos. In contrast tothe wild-type control embryo (FIG. 8J), which shows a low level ofbackground LacZ staining, the rRosa26 heterozygous rat embryo exhibitedubiquitous expression of the LacZ reporter throughout the embryo.

FIG. 9A-B provides a photograph showing the analysis of the chromosomenumber of the ACI.G1 rat ES cell line.

FIG. 10A-B provides a photograph showing the analysis of the chromosomenumber of the DA.2B rat ES cell line.

FIG. 11A-B provides a photograph showing the analysis of the chromosomenumber of the DA.C2 rat ES cell line.

DETAILED DESCRIPTION

The present methods and compositions now will be described more fullyhereinafter with reference to the accompanying drawings, in which some,but not all embodiments of the methods and compositions are shown.Indeed, these methods and compositions may be embodied in many differentforms and should not be construed as limited to the embodiments setforth herein; rather, these embodiments are provided so that thisdisclosure will satisfy applicable legal requirements. Like numbersrefer to like elements throughout.

Many modifications and other embodiments of the methods and compositionsset forth herein will come to mind to one skilled in the art to whichthis methods and compositions pertains having the benefit of theteachings presented in the foregoing descriptions and the associateddrawings. Therefore, it is to be understood that the methods andcompositions are not to be limited to the specific embodiments disclosedand that modifications and other embodiments are included within thescope of the appended claims. Although specific terms are employedherein, they are used in a generic and descriptive sense only and notfor purposes of limitation.

I. Overview

The rat has long been the preferred rodent model organism for severalfields of biomedical research such as cardiovascular disease,metabolism, toxicology, neurobiology and behavior. Hundreds of strainsof rat have been developed; some are excellent models for complex humandiseases such as hypertension, diabetes and cancer. However, progress inunderstanding the genetics of these models has been severely hampered bythe difficulty of modifying the rat genome in a controlled manner.Through the use of site-specific endonucleases it is possible to producemutations in a gene of interest, but this method remains imprecise andexpensive. Targeting and germline transmission of rat ES cells remains adifficult task to achieve.

Isolation of rat ES cells (rESC) from two inbred strains of rat isdescribed herein. rESC from the DA and ACI strains were derived. Thesecells express pluripotency markers and exhibit a normal 42X,Y karyotype.High percentage chimeras have been produced, by microinjection into SDhost embryos at the blastocyst stage, and transmission of the rESCgenome has been demonstrated through the germline for both strains.Using plasmid targeting vectors, we have produced targeted mutations inthe rat equivalent of the ROSA26 locus, and we have achieved germlinetransmission of the targeted allele in both strains. These heterozygousanimals express lacZ in all tissues at all stages examined.

In various aspects, ES cells were derived from the ACI strain in orderto obtain a favorable number of male progeny from ACI donor ES cells. Inone embodiment, the amount of male progeny is about 50%.

In various aspects, ES cells were derived from the DA strain in order toobtain primarily female progeny.

II. Rat Embryonic Stem (ES) Cells

Various compositions and methods are provided herein which compriseembryonic stem (ES) cells from rat. Stem cells are a cell populationpossessing the capability to self-renew indefinitely and arepluripotent. An “embryonic stem cell” or an “ES cell” comprises a stemcell obtained from an embryo or a fetus. The various rat ES cellsprovided herein can have one or more of any of the following properties:

(a) have germ-line competency, meaning when the rat ES cell is implantedinto a rat host embryo, the genome of the rat ES cell line istransmitted into an offspring;

(b) have germ-line competency following at least one targeted geneticmodification, meaning when the rat ES cell having the targeted geneticmodification is implanted into a rat host embryo, the targeted geneticmodification within the genome of the rat ES cell line is transmittedinto an offspring;

(c) have pluripotency in vitro;

(d) have totipotency in vitro;

(e) when cultured in vitro loosely adhere to a feeder cell layer;

(f) when cultured in vitro form sphere-like colonies when plated on afeeder cell layer in vitro;

(g) maintain pluripotency when cultured in vitro under conditionscomprising a feeder cell layer that is not genetically modified toexpress leukemia inhibitory factor (LIF), wherein the culture mediacomprises a sufficient concentration of LIF;

(h) maintain pluripotency when cultured in vitro under conditionscomprising a feeder cell layer, wherein the culture media comprisesmouse LIF or an active variant or fragment thereof;

(i) comprise a molecular signature that is characterized by

-   -   i) the expression of one or more of rat ES cell-specific genes        comprising Adherens Junctions Associate Protein 1 (Ajap1),        Claudin 5 (Cldn5), Cdc42 guanine nucleotide exchange factor 9        (Arhgef9), Calcium/calmodulin-dependent protein kinase IV        (Camk4), ephrin-A1 (Efna1), EPH receptor A4 (Epha4), gap        junction protein beta 5 (Gjb5), Insulin-like growth factor        binding protein-like 1 (Igfbpl1), Interleukin 36 beta (Il1f8),        Interleukin 28 receptor, alpha (Il28ra), left-right        determination factor 1 (Lefty1), Leukemia inhibitory factor        receptor alpha (Lifr), Lysophosphatidic acid receptor 2 (Lpar2),        Neuronal pentraxin receptor (Ntm), Protein tyrosine phosphatase        non-receptor type 18 (Ptpn18), Caudal type homeobox 2 (Cdx2),        Fibronectin type III and ankyrin repeat domains 1 (Fank1),        Forkhead box E1 (thyroid transcription factor 2) (Foxe1),        Hairy/enhancer-of-split related with YRPW motif 2 (Hey2),        Forkhead box E1 (thyroid transcription factor 2) (Foxe1),        Hairy/enhancer-of-split related with YRPW motif 2 (Hey2),        Lymphoid enhancer-binding factor 1 (Lef1), Sal-like 3        (Drosophila) (Sall3), SATB homeobox 1 (Satb1), miR-632, or a        combination thereof;    -   ii) the expression of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more        of the rat ES cell-specific genes comprising Adherens Junctions        Associate Protein 1 (Ajap1), Claudin 5 (Cldn5), Cdc42 guanine        nucleotide exchange factor 9 (Arhgef9),        Calcium/calmodulin-dependent protein kinase IV (Camk4),        ephrin-A1 (Efna1), EPH receptor A4 (Epha4), gap junction protein        beta 5 (Gjb5), Insulin-like growth factor binding protein-like 1        (Igfbpl1), Interleukin 36 beta (Il1f8), Interleukin 28 receptor,        alpha (Il28ra), left-right determination factor 1 (Lefty1),        Leukemia inhibitory factor receptor alpha (Lifr),        Lysophosphatidic acid receptor 2 (Lpar2), Neuronal pentraxin        receptor (Ntm), Protein tyrosine phosphatase non-receptor type        18 (Ptpn18), Caudal type homeobox 2 (Cdx2), Fibronectin type III        and ankyrin repeat domains 1 (Fank1), Forkhead box E1 (thyroid        transcription factor 2) (Foxe1), Hairy/enhancer-of-split related        with YRPW motif 2 (Hey2), Forkhead box E1 (thyroid transcription        factor 2) (Foxe1), Hairy/enhancer-of-split related with YRPW        motif 2 (Hey2), Lymphoid enhancer-binding factor 1 (Lef1),        Sal-like 3 (Drosophila) (Sall3), SATB homeobox 1 (Satb1),        miR-632, or a combination thereof;    -   iii) at least a 20-fold increase in the expression of one or        more of the rat ES cell-specific genes as set forth in Table 14        when compared to a F1H4 mouse ES cell;    -   iv) at least a 20-fold increase in the expression of at least 2,        3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25 or more of the rat ES cell-specific genes as        set forth in Table 14 when compared to a F1H4 mouse ES cell;    -   v) the expression of one or more of rat ES cell-specific genes        as set forth in Table 13;    -   vi) the expression of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,        40, 45, 50 or more of the rat ES cell-specific genes as set        forth in Table 13;    -   vii) at least a 20-fold increase in the expression of one or        more of the rat ES cell-specific genes as set forth in Table 13        when compared to a F1H4 mouse ES cell;    -   viii) at least a 20-fold increase in the expression of at least        2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,        20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more of the rat ES        cell-specific genes as set forth in Table 13 when compared to a        F1H4 mouse ES cell;    -   ix) at least a 20-fold decrease in the expression of one or more        of the rat ES cell-specific genes as set forth in Table 12 when        compared to a F1H4 mouse ES cell; and/or    -   x) at least a 20-fold decrease in the expression of at least 2,        3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more of the rat ES        cell-specific genes as set forth in Table 12 when compared to a        F1H4 mouse ES cell;    -   xi) any combination of expression of the rat ES cell-specific        genes of parts (i)-(x);    -   xii) a relative expression level of pluripotency markers as        shown in Table 15 for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17 or 18 of the listed pluripotency markers.        See, pluripotency ranking column of Table 15 for relative        expression levels;    -   xiii) a relative expression level of the mesodermal markers as        shown in Table 15 for at least 2, 3, or 4 of the listed        mesodermal markers. See, mesodermal ranking column in Table 15        for relative expression levels;    -   xiv) a relative expression level of endodermal markers as shown        in Table 15 for at least 2, 3, 4, 5, or 6 of the listed        endodermal markers. See, endodermal ranking column in Table 15        for relative expression levels;    -   xv) a relative expression level of neural markers as shown in        Table 15 for at least 2 and 3 of the listed neural markers. See,        neural ranking column in table 15 for relative expression        levels;    -   xvi) a relative expression level of trophectoderm markers as        shown in Table 15 for the listed trophectoderm markers. See,        trophectoderm ranking column in table 15 for relative expression        levels;    -   xvii) any relative expression level of one or more (2, 3, 4, 5,        6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,        23, 24, 25, 26, 27, 28, 29 or 30) of the pluripotency markers,        mesodermal markers, endodermal markers, neural markers and/or        trophectoderm markers set forth in Table 15;    -   xviii) the relative expression level of each of the markers set        forth in table 15;    -   xix) any combination of the signatures set forth in xii-xiix;        and/or    -   xx) any combination of the signature set forth in i-xiix;

(j) have the ability to produce a F0 rat;

(k) are capable of being subcultured and maintaining theundifferentiated state;

(l) have the same number of chromosomes as a normal rat cell;

(m) maintain pluripotency in vitro without requiring paracrine LIFsignaling; and/or

(n) have self renewal, meaning they divide indefinitely whilemaintaining pluripotency.

One or more of the characteristics outlined in (a)-(n) can be present inan isolated rat ES cell, a rat ES cell population or a rat ES cell lineprovided herein, wherein the rat ES cells have not undergone a targetedgenetic modification. In other embodiments, one or more of thecharacteristics outlined in (a)-(n) can be present in an isolated rat EScell, a rat ES cell population or a rat ES cell line provided hereinthat has one or more targeted genetic modifications. A targeted geneticmodification comprises an alteration in the genome of the rat ES celland includes, for example, an insertion, a deletion, a knockout, aknockin, a mutation, or a combination thereof. In other instances, thetargeted genetic modification comprises at least one insertion of aheterologous polynucleotide into the genome of the rat ES cell. Afurther description of such targeted genetic modifications are discussedelsewhere herein.

In specific embodiments, the various rat ES cells and cell linesprovided herein are germ-line competent, meaning when the rat ES cell isimplanted into a rat host embryo, the genome of the rat ES cell istransmitted into an offspring. Such transmission into the offspring(i.e., the F1 population) can occur when the rat ES cell has notundergone a targeted genetic modification. In addition, a rat ES cellhaving a targeted genetic modification are also germ-line competent,meaning when the rat ES cell having the targeted genetic modification isimplanted into a rat host embryo, the targeted genetic modification ofthe rat ES cell is transmitted to the offspring (i.e., the F1population.) Thus, in various aspects, the rat ES cells and methodsdescribed herein are employed to obtain a high frequency, or highefficiency, of germline transmission of a rat cell genome from both ratES cells that have not undergone a targeted genetic modification andalso from rat ES cells that have undergone a targeted geneticmodification. In various embodiments, the frequency of germlinetransmission is greater than 1:600, greater than 1:500, greater than1:400, greater than 1:300, greater than 1:200, and greater than 1:100.In various embodiments, the frequency of germline transmission isgreater than 1%, greater than 2%, greater than 3%, greater than 4%,greater than 5%, greater than 6%, greater than 7%, greater than 8%,greater than 9%, greater than 10%, up to about 16%, greater than 25%,greater than 50%, greater than 60%, greater than 65%, greater than 70%,greater than 75% or greater. In various embodiments, the frequency ofgermline transmission ranges from 9% to 16%. In various aspects, percentof donor rESC-derived progeny in the F1 generation is 1% or more, 2% ormore, 3% or more, 10% or more, 20% or more, 30% or more, 40% or more,50% or more, 60% or more, from 3% to about 10% or more; from 3% or moreto about 63%, from about 10% to about 30%, from about 10% to about 50%,from about 30% to about 70%, from about 30% to about 60%, from about 20%to about 40%, from about 20% to 65%, or from about 40% to 70%. Thus, arat ES cell provided herein that has not undergone a targeted geneticmodification or, alternatively, a rat ES cell that has a targetedgenetic modification have the ability to transmit their genome into theF1 population.

A rat ES cell that has not undergone a targeted genetic modification ora rat ES cell that has a targeted genetic modification can bepluripotent and/or totipotent. “Pluripotent” or “pluripotency” refers toa stem cell that has the potential to differentiate into any of thethree germ layers: endoderm, mesoderm, or ectoderm. Cell potency is ageneral term which describes a cell's ability to differentiate intoother cell types. See, for example, Hans et al. (2007). “The Potentialof Stem Cells: An Inventory”. Human biotechnology as Social Challenge.Ashgate Publishing, Ltd. p. 28, herein incorporate by reference. Theterm “totipotency” or “totipotent” is the ability of a single cell todivide and produce all of the differentiated cells in an organism. See,for example, Western P (2009). Int. J. Dev. Biol. 53 (2-3): 393-409,herein incorporated by reference. In specific embodiments, the variousES cells disclosed herein can be either pluripotent and/or totipotent.

Various methods can be used to determine if a rat ES cell ispluripotent. For example, the ES cell can be assayed for the expressionof various pluripotent markers including, but not limited to, Oct-4,Sox2, alkaline phosphatase, or a combination thereof. See, for example,Okamoto, K. et al., Cell, 60: 461-472 (1990), Scholer, H. R. et al.,EMBO J. 9: 2185-2195 (1990)) and Nanog (Mitsui, K. et al., Cell, 113:631-642 (2003), Chambers, I. et al., Cell, 113: 643-655 (2003) forvarious methods of assaying for the presence or the level of suchmarkers. See, also FIGS. 2 and 3 provided herein. Other pluripotencymarkers include, for example, the presence of at least 1, 2, 3, 4, or 5pluripotency marker comprising Nanog, Klf4, Dppa2, Fgf4, Rex1, Eras,Err-beta and/or Sall3. Other pluripotency markers include, for example,the absence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 pluripotencymarker comprising T/Brachyury, Flk1, Nodal, Bmp4, Bmp2, Gata6, Sox17,Hhex1, Sox7, and/or Pax6.

In specific embodiments, the expression and/or the level of expressionof these markers can be determined using RT-PCR. Various kits areavailable to determine the level and/or presence of alkalinephosphatase, including, for example, an ALP tissue staining kit (Sigma)and Vector Red Alkaline Phosphatase Substrate Kit I (Funakoshi) and thelike. Additional assays include in situ hybridization,immunohistochemistry, immunofluorescence. In specific embodiments, therat ES cell is characterized by expression of at least one pluripotencymarker, including for example expression of Oct-4, Sox2, alkalinephosphatase, or a combination thereof, and preferably all three of thesemarkers.

The various rat ES cell provided herein (i.e. rat ES cells that have notundergone a targeted genetic modification and/or rat ES cells that havea targeted genetic modification) are capable of maintaining pluripotencyand/or totipotency while being maintained in in vitro culturingconditions. Thus, the various rat ES cells provide herein can, in someembodiments, be subcultured while still maintaining the undifferentiatedstate. Various methods of culturing the rat ES cells are discussed infurther detail elsewhere herein.

The rat embryonic stem cells provided herein have been isolated from therat embryo employing various isolation, purification, and cultureexpansion techniques which are discussed in detail elsewhere herein. Theterm “cell” as used herein refers to individual cells, cell lines, orcultures derived from such cells. An “isolated” rat ES cell or ratembryo has been removed from its natural environment. The term“isolated” can mean free from 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%of the constituents with which a component is found in its naturalstate. As used herein, a rat ES “cell line” comprises a population ofisolated rat cells that were developed from a single rat ES cell andtherefore the population of cells within a given cell line have auniform genetic makeup other than for mutations or karyotypic changesoccurring during propagation or during targeted genetic modifications.For example, as indicated elsewhere, the disclosed rat ES cells arecharacterized by a high level of euploidy. Nevertheless, in some celllines the level of euploidy is less than 100% due to karyotypic changesin propagation of the line from a single cell. Moreover, a givenpopulation of rat ES cells can comprise at least 10 exp 3, 10 exp4,10×10⁴, 10×10⁵, 10×10⁶, 10×10⁷, 10×10⁸, 10×10⁹, or 10×10¹⁰ cells orgreater. Some cell populations have sufficient cells to permit selectionof a desired modified cell but not an excessively greater number so asto reduce the possibility of mutations or karyotypic changes developingin the cell line. For example, some cell populations have 10exp3 to10exp6 cells.

As discussed elsewhere herein, various methods are provided for thetargeted genetic modification of a rat ES cell line. When such methodsare carried out, at least one cell within a rat ES cell line containsthe targeted genetic modification. Through various culturing and/orselection techniques rat ES cell lines having one or more desiredtargeted genetic modifications are produced.

In specific embodiments, a rat ES cell, a population of rat ES cell or arat ES cell line (that have not undergone a targeted geneticmodification and/or have a targeted genetic modification) are euploid,and thus have a chromosome number that is an exact multiple of thehaploid number. In further embodiment, a rat ES cell, a population ofrat ES cells or a rat ES cell line (that have not undergone a targetedgenetic modification and/or have a targeted genetic modification) arediploid, and thus have two haploid sets of homologous chromosomes. Whenreferring to a rat ES cell population or a population of cells from agiven population of rat ES cells or a rat ES cell line (that have notundergone a targeted genetic modification and/or have a targeted geneticmodification), at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%of the cells with the given population or euploid and/or diploid. Inother instances, when referring to a rat ES cell population or apopulation of cells from a given rat ES cell line (that have notundergone a targeted genetic modification and/or have a targeted geneticmodification), at least about 50% to 95%, about 60% to 90%, about 60% to95%, about 60% to 85%, about 60% to 80%, about 70% to 80%, about 70% to85%, about 70% to about 90%, about 70% to about 95%, about 70% to about100%, about 80% to about 100%, about 80% to about 95%, about 80% toabout 90%, about 90% to about 100%, about 90% to about 99%, about 90% toabout 98%, about 90% to about 97%, about 90% to about 95% of the cellswithin the given population are euploid and/or diploid.

In still further embodiments, a rat ES cell, a population of rat EScells or a rat ES cell line (that have not undergone a targeted geneticmodification and/or have a targeted genetic modification) have 42chromosomes. When referring to a rat ES cell population or a populationof cells from a given rat ES cell line (that have not undergone atargeted genetic modification and/or have a targeted geneticmodification) at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%of the cells with the given population have 42 chromosomes. In otherinstances, when referring to a rat ES cell population or a population ofcells from a given rat ES cell line (that have not undergone a targetedgenetic modification and/or have a targeted genetic modification) atleast about 50% to 95%, about 60% to 90%, about 60% to 95%, about 60% to85%, about 60% to 80%, about 70% to 80%, about 70% to 85%, about 70% toabout 90%, about 70% to about 95%, about 70% to about 100%, about 80% toabout 100%, about 80% to about 95%, about 80% to about 90%, about 90% toabout 100%, about 90% to about 99%, about 90% to about 98%, about 90% toabout 97%, about 90% to about 95% of the cells within the givenpopulation have 42 chromosomes.

In further embodiments, a rat ES cell, a population of rat ES cells or arat ES cell line (that have not undergone a targeted geneticmodification and/or have a targeted genetic modification) providedherein form sphere-like colonies when plated on a feeder cell layer invitro. The “sphere-like” morphology refers to the shape of rat ES cellcolonies in culture, rather than the shape of individual ES cells. Therat ES cell colonies are spherical-like. Colonies which are looselyattached to the feeder cells appear circular (have a circular-likemorphology). Free-floating colonies are spherical-like. The rat ES cellcolonies are spherical-like and very compact, meaning: the boundariesbetween cells are very hard to see. The edge of the colony appearsbright and sharp. Individual nuclei are difficult to distinguish becausethe cells are very small (so that the nucleus takes up most of thevolume of the cell). Mouse ES Cells form elongated colonies and attachstrongly to feeder cells. mESC morphology can vary with strain; e.g. B6colonies are rounder and more domed than F1H4 colonies but are stillmore elongated than rESC. Human ES cell colonies are flatter and morespread out than mESC colonies. The instant rat ES colonies are not flatand do not resemble human ES cell colonies.

In still further embodiments, a rat ES cell, a population of rat EScells or a rat ES cell line (that have not undergone a targeted geneticmodification and/or have a targeted genetic modification) have acircular morphology. A morphology scale for a circle is provided below,where a score of a 10 represents a perfect circle and a score of a 1represents an ellipse.

Morphology scale of a circle:

-   -   10=A circle with a structure having a longitudinal axis and a        vertical axis that run through the center of the structure and        are of equal length.    -   9=A structure having a longitudinal axis and vertical axis that        run through the center of the structure, wherein one of the axis        is between 0.9999 to 0.9357 the length of the other axis.    -   8=A structure having a longitudinal axis and vertical axis that        run through the center of the structure, wherein one of the axis        is between 0.9357 to 0.875 the length of the other axis.    -   7=A structure having a longitudinal axis and vertical axis that        run through the center of the structure, wherein one of the axis        is between 0.875 to about 0.8125 the length of the other axis.    -   6=A structure having a longitudinal axis and vertical axis that        run through the center of the structure, wherein one of the axis        is between 0.8125 to 0.750 the length of the other axis.    -   5=A structure having a longitudinal axis and vertical axis that        run through the center of the structure, wherein one of the axis        is between 0.750 to 0.6875 the length of the other axis.    -   4=A structure having a longitudinal axis and vertical axis that        run through the center of the structure, wherein one of the axis        is between 0.6875 to 0.625 the length of the other axis.    -   3=A structure having a longitudinal axis and vertical axis that        run through the center of the structure, wherein one of the axis        is between 0.625 to 0.5625 the length of the other axis.    -   2=A structure having a longitudinal axis and vertical axis that        run through the center of the circle, wherein one of the axis is        between 0.5625 to 0.523 the length of the other axis.    -   1=An ellipse is defined as having a longitudinal axis and        vertical axis that run through the center of the structure,        wherein one of the axis is between 0.523 to 0.500 the length of        the other axis.

In one non-limiting embodiment, the rat ES cell population or apopulation of cells from a given rat ES cell line (that have notundergone a targeted genetic modification and/or have a targeted geneticmodification) have at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% of the cells with the given population have a circular morphologyscore of a 10, 9 or 8. In other embodiments, the rat ES cell populationor a population of cells from a given rat ES cell line (that have notundergone a targeted genetic modification and/or have a targeted geneticmodification) have at least about 50% to 95%, about 60% to 90%, about60% to 95%, about 60% to 85%, about 60% to 80%, about 70% to 80%, about70% to 85%, about 70% to about 90%, about 70% to about 95%, about 70% toabout 100%, about 80% to about 100%, about 80% to about 95%, about 80%to about 90%, about 90% to about 100%, about 90% to about 99%, about 90%to about 98%, about 90% to about 97%, about 90% to about 95% of thecells within the given population have a circular morphology score of a10, 9, or 8.

In another non-limiting embodiment, the rat ES cell population or apopulation of cells from a given rat ES cell line (that have notundergone a targeted genetic modification and/or have a targeted geneticmodification) have at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% of the cells with the given population have a circular morphologyscore of a 7, 6, 5, 4 or 3. In other non-limiting embodiments, the ratES cell population or a population of cells from a given rat ES cellline (that have not undergone a targeted genetic modification and/orhave a targeted genetic modification) have at least about 50% to 95%,about 60% to 90%, about 60% to 95%, about 60% to 85%, about 60% to 80%,about 70% to 80%, about 70% to 85%, about 70% to about 90%, about 70% toabout 95%, about 70% to about 100%, about 80% to about 100%, about 80%to about 95%, about 80% to about 90%, about 90% to about 100%, about 90%to about 99%, about 90% to about 98%, about 90% to about 97%, about 90%to about 95% of the cells within the given population have a circularmorphology score of a 7, 6, 5, 4, or 3.

In still further embodiments, sphere-like colonies form when the rat EScells (that have not undergone a targeted genetic modification and/orhave a targeted genetic modification) are plated on a feeder cell layerin vitro. A morphology scale for a sphere is provided below, where ascore of a 10 represents a perfect sphere and a score of a 1 representsa three dimensional elliptical structure.

Morphology scale of a sphere-like structure:

-   -   10=A sphere is a structure having an X-axis and a Y-axis and a        Z-axis each of which runs through the center of the structure        and are of equal length.    -   9=A structure having an X axis and a Y-axis and a Z-axis that        run through the center of the structure, wherein one of the axis        is between 0.9999 to 0.9357 the length of at least one of the        other axes.    -   8=A structure having an X axis and a Y-axis and a Z-axis that        run through the center of the structure, wherein one of the axis        is between 0.9357 to 0.875 the length of at least one or both of        the other axes.    -   7=A structure having an X axis and a Y-axis and a Z-axis that        run through the center of the structure, wherein one of the axis        is between 0.875 to 0.8125 the length of at least one or both of        the other axes.    -   6=A structure having an X axis and a Y-axis and a Z-axis that        run through the center of the structure, wherein one of the axis        is between 0.8125 to 0.750 the length of at least one or both of        the other axes.    -   5=A structure having an X axis and a Y-axis and a Z-axis that        run through the center of the structure, wherein one of the axis        is 0.750 to 0.6875 the length of at least one or both of the        other axes.    -   4=A structure having an X axis and a Y-axis and a Z-axis that        run through the center of the structure, wherein one of the axis        is 0.6875 to 0.625 the length of at least one or both of the        other axes.    -   3=A structure having an X axis and a Y-axis and a Z-axis that        run through the center of the structure, wherein one of the axis        is between 0.625 to 0.5625 the length of at least one or both of        the other axes.    -   2=A structure having an X axis and a Y-axis and a Z-axis that        run through the center of the structure, wherein one of the axis        is between 0.5625 to 0.523 the length of at least one or both of        the other axes.    -   1=A structure having an X axis and a Y-axis and a Z-axis that        run through the center of the structure, wherein one of the axis        is between 0.523 to 0.500 the length of at least one or both of        the other axes.

In one non-limiting embodiment, the rat ES cell population or apopulation of cells from a given rat ES cell line (that have notundergone a targeted genetic modification and/or have a targeted geneticmodification) have at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% of the colonies that form when the cells are plated on a feedercell layer in vitro have a sphere-like morphology of a 10, 9 or 8. Inother embodiments, the rat ES cell population or a population of cellsfrom a given rat ES cell line (that have not undergone a targetedgenetic modification and/or have a targeted genetic modification) haveat least about 50% to 95%, about 60% to 90%, about 60% to 95%, about 60%to 85%, about 60% to 80%, about 70% to 80%, about 70% to 85%, about 70%to about 90%, about 70% to about 95%, about 70% to about 100%, about 80%to about 100%, about 80% to about 95%, about 80% to about 90%, about 90%to about 100%, about 90% to about 99%, about 90% to about 98%, about 90%to about 97%, about 90% to about 95% of the colonies that form when thecells are plated on a feeder cell layer in vitro have a sphere-likemorphology of a 10, 9 or 8.

In another non-limiting embodiment, the rat ES cell population or apopulation of cells from a given rat ES cell line (that have notundergone a targeted genetic modification and/or have a targeted geneticmodification) have at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% of the colonies that form when the cells are plated on a feedercell layer in vitro have a sphere-like morphology of a 7, 6, 5, 4, or 3.In other embodiments, the rat ES cell population or a population ofcells from a given rat ES cell line (that have not undergone a targetedgenetic modification and/or have a targeted genetic modification) haveat least about 50% to 95%, about 60% to 90%, about 60% to 95%, about 60%to 85%, about 60% to 80%, about 70% to 80%, about 70% to 85%, about 70%to about 90%, about 70% to about 95%, about 70% to about 100%, about 80%to about 100%, about 80% to about 95%, about 80% to about 90%, about 90%to about 100%, about 90% to about 99%, about 90% to about 98%, about 90%to about 97%, about 90% to about 95% of the colonies that form when thecells are plated on a feeder cell layer in vitro have a sphere-likemorphology of a 7, 6, 5, 4, or 3.

A given rat ES cell, a population of rat ES cells or a rat ES cell lineprovided herein can be a male (XY) rat ES cell, a male (XY) populationof rat ES cells, or a male (XY) rat ES cell line. In other embodiments,a population of rat ES cells or a rat ES cell line provided herein canbe a female (XX) rat ES cell, a female (XX) population of rat ES cells,or a female (XX) rat ES cell line. Any such rat ES cell, population ofrat ES cells or rat ES cell line can comprise the euploidy and/ordiploidy as described above.

The various rat ES cell provided herein can be from any rat strain,including but not limited to, an ACI rat strain, a Dark Agouti (DA) ratstrain, a Wistar rat strain, a LEA rat strain, a Sprague Dawley (SD) ratstrain, or a Fischer rat strain such as Fisher F344 or Fisher F6. Thevarious rat ES cells can also be obtained from a strain derived from amix of two or more strains recited above. In one embodiment, the rat EScell is derived from a strain selected from a DA strain and an ACIstrain. In a specific embodiment, the rat ES cell is derived from an ACIstrain. The ACI rat strain is are characterized as having black agouti,with white belly and feet and an RTI^(av1) haplotype. Such strains areavailable from a variety of sources including Harlan Laboratories. Inother embodiments, the various rat ES cells are from a Dark Agouti (DA)rat strain which is characterized as having an agouti coat and anRTI^(av1) haplotype. Such rats are available from a variety of sourceincluding Charles River and Harlan Laboratories. In a furtherembodiment, the various rat ES cells provided herein are from an inbredrat strain.

In specific embodiments the rat ES cell line is from an ACI rat andcomprises the ACI.G1 rat ES cell as described herein. In anotherembodiment, the rat ES cell line is from a DA rat and comprises theDA.2B rat ES cell line or the DA.2C rat ES cell line as describedherein. A given rat ES cell provided herein can be obtained from a ratembryo at any stage of rat embryo development. Representative stages ofrat embryo development are outline below in Table 1. The rat embryosemployed to derive the rat ES cells can be a morula-stage embryo, ablastocyst-stage embryo, or a rat embryo at a developmental stagebetween a morula-stage embryo and a blastocyst-stage embryo. Thus, inspecific embodiments, the rat embryo employed is at or between theWitschi stages of 5 and 7. In other embodiments, the rat embryo employedis at the Witschi stage 5, 6, or 7.

In one embodiment, the rat ES cell is obtained from a rat blastocyst. Inother embodiments, the rat ES cell is obtained from a blastocyst from asuperovulated rat. In other embodiments, the rat ES cells are obtainedfrom an 8-cell stage embryo which is then cultured in vitro until itdevelops into a morula-stage, blastocyst stage, an embryo between theWitschi stages 5 and 7, or into an embryo at the Witschi stage 5, 6, or7. At which time the embryos are then plated. Morula-stage embryoscomprise a compact ball of cells with no internal cavity.Blastocyst-stage embryos have a visible internal cavity (the blastocoel)and contain an inner cell mass (ICM). The ICM cells form ES cells.

TABLE 1 Stages of Rat Embryo Development Standard Stages Age (Witschi)(days) Identification of Stages Cleavage and Blastula 1 1 1 cell (inoviduct) 2 2 2 cells (in oviduct) 3 3 4 cells (in oviduct) 4 3.25 8-12cells (in oviduct) 5 3.5 Morula (in uterus) 6 4 Early blastocyst (inuterus) 7 5 Free blastocyst (in uterus) Gastrula 8 6 Implantingblastocyst, with trophoblastic cone and inner cell mass; outgrowth ofendoderm (hypoblast) 9 6.75 Diplotrophoblast; inner cell mass (pendant),covered with endoderm 10 7.25 Near complete implantation; pendant beginsdifferentiation into embryonic and extra-embryonic parts 11 7.75Completion of implantation; primary amniotic cyst; ectoplacental cone

Further provided are various rat ES cells (that have not undergone atargeted genetic modification and/or have a targeted geneticmodification) which are characterized by The rat ES cell ischaracterized by:

i) the expression of one or more of rat ES cell-specific genescomprising Adherens Junctions Associate Protein 1 (Ajap1), Claudin 5(Cldn5), Cdc42 guanine nucleotide exchange factor 9 (Arhgef9),Calcium/calmodulin-dependent protein kinase IV (Camk4), ephrin-A1(Efna1), EPH receptor A4 (Epha4), gap junction protein beta 5 (Gjb5),Insulin-like growth factor binding protein-like 1 (Igfbpl1), Interleukin36 beta (Il1f8), Interleukin 28 receptor, alpha (Il28ra), left-rightdetermination factor 1 (Lefty1), Leukemia inhibitory factor receptoralpha (Lifr), Lysophosphatidic acid receptor 2 (Lpar2), Neuronalpentraxin receptor (Ntm), Protein tyrosine phosphatase non-receptor type18 (Ptpn18), Caudal type homeobox 2 (Cdx2), Fibronectin type III andankyrin repeat domains 1 (Fank1), Forkhead box E1 (thyroid transcriptionfactor 2) (Foxe1), Hairy/enhancer-of-split related with YRPW motif 2(Hey2), Forkhead box E1 (thyroid transcription factor 2) (Foxe1),Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Lymphoidenhancer-binding factor 1 (Lef1), Sal-like 3 (Drosophila) (Sall3), SATBhomeobox 1 (Satb1), miR-632, or a combination thereof;

ii) the expression of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more of the rat EScell-specific genes comprising Adherens Junctions Associate Protein 1(Ajap1), Claudin 5 (Cldn5), Cdc42 guanine nucleotide exchange factor 9(Arhgef9), Calcium/calmodulin-dependent protein kinase IV (Camk4),ephrin-A1 (Efna1), EPH receptor A4 (Epha4), gap junction protein beta 5(Gjb5), Insulin-like growth factor binding protein-like 1 (Igfbpl1),Interleukin 36 beta (Il1f8), Interleukin 28 receptor, alpha (Il28ra),left-right determination factor 1 (Lefty1), Leukemia inhibitory factorreceptor alpha (Lifr), Lysophosphatidic acid receptor 2 (Lpar2),Neuronal pentraxin receptor (Ntm), Protein tyrosine phosphatasenon-receptor type 18 (Ptpn18), Caudal type homeobox 2 (Cdx2),Fibronectin type III and ankyrin repeat domains 1 (Fank1), Forkhead boxE1 (thyroid transcription factor 2) (Foxe1), Hairy/enhancer-of-splitrelated with YRPW motif 2 (Hey2), Forkhead box E1 (thyroid transcriptionfactor 2) (Foxe1), Hairy/enhancer-of-split related with YRPW motif 2(Hey2), Lymphoid enhancer-binding factor 1 (Lef1), Sal-like 3(Drosophila) (Sall3), SATB homeobox 1 (Satb1), miR-632, or a combinationthereof;

iii) at least a 20-fold increase in the expression of one or more of therat ES cell-specific genes as set forth in Table 14 when compared to aF1H4 mouse ES cell;

iv) at least a 20-fold increase in the expression of at least 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25 or more of the rat ES cell-specific genes as set forth in Table14 when compared to a F1H4 mouse ES cell;

v) the expression of one or more of rat ES cell-specific genes as setforth in Table 13;

vi) the expression of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 ormore of the rat ES cell-specific genes as set forth in Table 13;

vii) at least a 20-fold increase in the expression of one or more of therat ES cell-specific genes as set forth in Table 13 when compared to aF1H4 mouse ES cell;

viii) at least a 20-fold increase in the expression of at least 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 30, 35, 40, 45, 50 or more of the rat ES cell-specific genes asset forth in Table 13 when compared to a F1H4 mouse ES cell;

ix) at least a 20-fold decrease in the expression of one or more of therat ES cell-specific genes as set forth in Table 12 when compared to aF1H4 mouse ES cell; and/or

x) at least a 20-fold decrease in the expression of at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 30, 35, 40, 45, 50 or more of the rat ES cell-specific genes as setforth in Table 12 when compared to a F1H4 mouse ES cell;

xi) any combination of expression of the rat ES cell-specific genes ofparts (i)-(x);

xii) a relative expression level of pluripotency markers as shown inTable 15 for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17 or 18 of the listed pluripotency markers. See, pluripotencyranking column of Table 15 for relative expression levels;

xiii) a relative expression level of the mesodermal markers as shown inTable 15 for at least 2, 3, or 4 of the listed mesodermal markers. See,mesodermal ranking column in Table 15 for relative expression levels;

xiv) a relative expression level of endodermal markers as shown in Table15 for at least 2, 3, 4, 5, or 6 of the listed endodermal markers. See,endodermal ranking column in Table 15 for relative expression levels;

xv) a relative expression level of neural markers as shown in Table 15for at least 2 and 3 of the listed neural markers. See, neural rankingcolumn in table 15 for relative expression levels;

xvi) a relative expression level of trophectoderm markers as shown inTable 15 for the listed trophectoderm markers. See, trophectodermranking column in table 15 for relative expression levels;

xvii) any relative expression level of one or more (2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29 or 30) of the pluripotency markers, mesodermal markers,endodermal markers, neural markers and/or trophectoderm markers setforth in Table 15;

xviii) the relative expression level of each of the markers set forth intable 15;

xix) any combination of the signatures set forth in xii-xiix; and/or

xx) any combination of the signature set forth in i-xiix.

In one embodiment, when transplanted into a pre-morula stage rat embryo,the rat ES cell (that have not undergone a targeted genetic modificationand/or have a targeted genetic modification) can contribute to at least90% of the cells in an F0 generation, contribute to at least 95% of thecells in an F0 generation, contribute to at least 96% of the cells in anF0 generation, contribute to at least 97% of the cells in an F0generation, contribute to at least 98% of the cells in an F0 generation,or contribute to at least 99% of the cells in an F0 generation.

III. Derivation and Propagation of Rat Embryonic Stem (ES) Cells

Various methods are provided for obtaining the rat ES cells disclosedherein. In specific embodiments, such methods comprise (a) providing anin vitro culture comprising a feeder cell layer and a population ofisolated rat embryonic stem (ES) cells; (b) culturing in vitro underconditions which are sufficient to maintain pluipotency and/ortotipotency of the isolated rat ES cell. Such methods thereby allow forthe propagation of a rat ES cell population and/or a rat ES cell line.

In one embodiment, a method for culturing a rat embryonic stem cell lineis provided. Such methods comprises culturing in vitro a feeder celllayer and a rat ES cell line, wherein the culture conditions maintainpluripotency of the rat ES cells and comprise a media having mouseleukemia inhibitory factor (LIF) or an active variant or fragmentthereof. The various methods further comprise passaging and culturing invitro the cells of the rat ES cell line, wherein each subsequent invitro culturing comprises culturing the rat ES cells on the feeder celllayer under conditions that maintain pluripotency of the rat ES cellsand comprises a media having mouse LIF or an active variant or fragmentthereof.

i. Culture Conditions

The culture media employed in the various methods and compositions willmaintain the rat ES cells. The terms “maintaining” and “maintenance”refer to the stable preservation of at least one or more of thecharacteristics or phenotypes of the rat ES cells outline herein. Suchphenotypes can include maintaining pluripotency and/or totipotency, cellmorphology, gene expression profiles and the other functionalcharacteristics of the rat stem cells described herein. The term“maintain” can also encompass the propagation of stem cells, or anincrease in the number of stem cells being cultured. The term furthercontemplates culture conditions that permit the stem cells to remainpluripotent, while the stem cells may or may not continue to divide andincrease in number.

The term “feeder cell” or “feeder cell layer” refers to a culture ofcells that grow in vitro and secrete at least one factor into theculture medium that is used to support the growth of another cell ofinterest in the culture. The feeder cells employed herein aid inmaintaining the pluripotency of the rat ES cells, and in specificembodiments, one or more of the other characteristics or phenotypesdescribed herein. Various feeder cells can be used including, forexample, mouse embryonic fibroblasts, including mouse embryonicfibroblasts obtained between the 12^(th) and 16^(th) day of pregnancy.In specific embodiments, feeder cell layer comprises a monolayer ofmitotically inactivated mouse embryonic fibroblasts (MEFs).

The in vitro cultures of the rat ES cells further comprise an effectiveamount of Leukemia Inhibitory Factor (LIF) or an active variant orfragment thereof. Leukemia inhibitory factor (LIF) belongs to the IL-6receptor family. LIF binds to a heterodimeric membrane receptor made upof a LIF-specific subunit, gp190 or LIFR, and the subunit gp130, whichis shared with the other members of the IL-6 family. LIF inhibits thedifferentiation of embryonic stem cells in mice and contribute to stemcell self-renewal. Human and mouse LIF share 79% sequence homology andexhibit cross-species activity. Rat LIF (rtLIF) is a 22.1 kDa proteincontaining 202 amino acid residues that exhibits 91% amino acid sequenceidentity with murine LIF (Takahama et al. 1998). There are six possibleasparagine-linked glycosylation (N-glycosylation) sites which areconserved among the LIF polypeptide from the various species and anadditional site of Asn150 which is specific for rat LIF. The tertiarystructure of the mouse LIF and its function is described in furtherdetail in Aikawa et al. (1998) Biosci. Biotechnol. Biochem. 62 1318-1325and Senturk et al. (2005) Immunology of Pregnancy, editor Gil Mor., U.S.Pat. No. 5,750,654 and D P Gearing (1987) EMBO Journal 1987-12-20, eachof which is herein incorporated by reference in their entirety. Apartial mouse LIF sequence is reported on the SwissProt website underthe accession number P09056.

Mouse LIF activity is assessed by its ability to induce differentiationof M1 myeloid leukemia cells. The specific activity is 1×10⁶ units/ml(Cat. No. 03-0011 from Stemgent) and 1×10⁷ units/ml (Cat. No.03-0011-100 from Stemgent), where 50 units is defined as the amount ofmouse LIF required to induce differentiation in 50% of the M1 coloniesin 1 ml of medium. See, also, Williams, R. L. et al. (1988) Nature 336:684-687; Metcalf, D. et al. (1988) Leukemia 2: 216-221; Niwa, H. et al.(2009) Nature 460: 118-122; Xu, J. et al. (2010) Cell Biol Int. 34:791-797; Fukunaga, N. et al. (2010) Cell Reprogram. 12: 369-376; and,Metcalf D. (2003) Stem Cells 21: 5-14, each of which is hereinincorporated by reference in their entirety. An “effective amount ofLIF” comprises a concentration of LIF that allows the rat ES cells of anin vitro culture to remain in an undifferentiated pluripotent state.Various markers that can be used to assay for the cells remaining in apluripotent state are discussed elsewhere herein.

The LIF polypeptide employed in the various methods and compositionsprovided herein can be from any organism, including from a mammal, arodent, a human, a rat or a mouse. In one embodiment, the LIFpolypeptide is from a mouse. In still further embodiments, the mouse LIFpolypeptide comprises the amino acid sequence set forth in SwissProtAccession number: P09056, which is herein incorporated by reference inits entirety and is also set forth in SEQ ID NO: 1.

In other embodiments, an active variant or fragment of the mouse LIFpolypeptide as set forth in SEQ ID NO: 1 or in SwissProt Accessionnumber: P09056 can be used. Such active variants and fragments(including active variants having at least 75%, 80%, 85% 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1)are discussed in further detail elsewhere herein.

LIF polypeptide or the active variant or fragment thereof can beprovided to the in vitro culture in a variety of ways. In oneembodiment, the effective amount of the LIF polypeptide or the activevariant or fragment thereof is added to the culture media. In otherembodiments, the feeder cells have been genetically modified tooverexpress the LIF polypeptide or the active variant or fragmentthereof. Such feeder cells include feeder cells prepared fromgamma-irradiated or mitomycin-C treated DIA-M mouse fibroblasts thatexpress matrix-associated LIF. Method of generating and using suchgenetically modified feeder cells can be found, for example, in See,Buehr et al. (2003) Biol Reprod 68:222-229, Rathj en et al. (1990) Cell62 1105-1115, and Buehr et al. (2008) Cell 135:1287-1298, each of whichis herein incorporated by reference. The heterologous LIF expressed inthe feeder cells can be from the same organism as the feeder cells orfrom an organism that is different from that of the feeder cell. Inaddition, the heterologous LIF expressed in the feeder cells can be fromthe same or from a different organism than the ES cells the feeder layeris supporting.

In still other embodiments, the feeder cells employed in the variousmethods disclosed herein are not genetically modified to express aheterologous LIF polypeptide or an active variant or fragment thereof.Thus, in particular embodiments, the monolayer of mitoticallyinactivated mouse embryonic fibroblast employed in the methods has notbeen genetically modified to express a heterologous LIF polypeptide.

In other embodiments, the LIF polypeptide or the active variant orfragment thereof is added to the culture media. When LIF is added to theculture media, the LIF can be from any organism, including from amammal, a rodent, a human, a rat or a mouse. In one embodiment, the LIFpresent in the culture media is from a mouse. In still furtherembodiments, the mouse LIF polypeptide comprises the amino acid sequenceset forth in SEQ ID NO:1. In other embodiments, an active variant orfragment of the mouse LIF polypeptide as set forth in SEQ ID NO:1 can beused. Such active variants and fragments (including active variantshaving at least 75%, 80%, 85% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity to SEQ ID NO: 1) are discussed in furtherdetail elsewhere herein.

In specific embodiments, the rat ES cells and rat ES cell lines providedherein maintain pluripotency in vitro without requiring paracrine LIFsignaling.

In specific embodiments, LIF or an active variant or fragment thereof ispresent in the culture media at any concentration that maintains the ratES cells. LIF polypeptide or active variant or fragment thereof ispresent in the culture media at about 25 U/ml to about 50 U/ml, at about50 U/ml to about 100 U/ml, at about 100 U/ml to about 125 U/ml, at about125 U/ml to about 150 U/ml, at about 150 U/ml to about 175 U/ml, atabout 175 U/ml to about 200 U/ml, at about 200 U/ml to about 225 U/ml,at about 225 U/ml to about 250 U/ml, at about 250 U/ml to about 300U/ml, to about 300 U/ml to about 325 U/ml, at about 325 U/ml to about350 U/ml, at about 350 U/ml to about 400 U/ml, at about 400 U/ml toabout 425 U/ml, at about 425 U/ml to about 450 U/ml, at about 450 U/mlto about 475 U/ml, at about 475 U/ml to about 500 U/ml, at about 75 U/mlto about 500 U/ml or greater. In other embodiments, LIF polypeptide oractive variant or fragment thereof is present in the culture media atabout 25 U/ml to about 50 U/ml, at about 25 U/ml to about 100 U/ml, atabout 75 U/ml to about 125 U/ml, at about 50 U/ml to about 150 U/ml, atabout 90 U/ml to about 125 U/ml, at about 90 U/ml to about 110 U/ml, atabout 80 U/ml to about 150 U/ml, at about 80 U/ml to about 125 U/ml. Ina specific embodiment, LIF polypeptide or active variant or fragmentthereof is present in the culture media at about 100 U/ml.

When mouse LIF is employed, the mouse LIF polypeptide or active variantor fragment thereof is present in the culture media at any concentrationthat maintains the rat ES cells. Mouse LIF polypeptide or active variantor fragment thereof is present at about 25 U/ml to about 50 U/ml, atabout 50 U/ml to about 100 U/ml, at about 100 U/ml to about 125 U/ml, atabout 125 U/ml to about 150 U/ml, at about 150 U/ml to about 175 U/ml,at about 175 U/ml to about 200 U/ml, at about 200 U/ml to about 225U/ml, at about 225 U/ml to about 250 U/ml, at about 250 U/ml to about300 U/ml, to about 300 U/ml to about 325 U/ml, at about 325 U/ml toabout 350 U/ml, at about 350 U/ml to about 400 U/ml, at about 400 U/mlto about 425 U/ml, at about 425 U/ml to about 450 U/ml, at about 450U/ml to about 475 U/ml, at about 475 U/ml to about 500 U/ml, at about 75U/ml to about 500 U/ml or greater. In other embodiments, mouse LIFpolypeptide or active variant or fragment thereof is present at about 25U/ml to about 50 U/ml, at about 25 U/ml to about 100 U/ml, at about 75U/ml to about 125 U/ml, at about 50 U/ml to about 150 U/ml, at about 90U/ml to about 125 U/ml, at about 90 U/ml to about 110 U/ml, at about 80U/ml to about 150 U/ml, at about 80 U/ml to about 125 U/ml. In aspecific embodiment, mouse LIF polypeptide or active variant or fragmentthereof is present in the culture media at about 100 U/ml.

The culture media employed maintains rat ES cells. As such, in specificembodiments, the culture media employed in the various method andcompositions will maintain the pluripotency of all or most of (i.e.,over 50%) of the rat ES cells in a cell line for a period of a at least5, 10 or 15 passages. In one embodiment, the culture media comprises oneor more compounds that assist in maintaining pluripotency. In oneembodiment, the culture media comprises a MEK pathway inhibitor and aglycogen synthase kinase-3 (GSK-3) inhibitor. The media can furthercomprise additional components that aid in maintaining the ES cells,including for example, FGF receptor inhibitors, ROCK inhibitors, and/orALK (TGFb receptor) inhibitors. A non-limiting example of an FGFreceptor inhibitors includes PD184352. A non-limiting example of a ROCKinhibitor includes Y-27632, and non-limiting example of an ALK (TGFbreceptor) inhibitor includes A-83-01. In specific embodiments, 2i media(table 2) is used with 10 uM ROCKi when thawing cryopreserved rESC orwhen re-plating rESC after dissociation with trypsin.

In other embodiments, the media comprises a combination of inhibitorsconsisting of a MEK pathway inhibitor and a glycogen synthase kinase-3(GSK-3) inhibitor. In one non-limiting embodiment, the culture mediacomprises a GSK-3 inhibitor comprising CHIR99021 and/or comprises a MEKinhibitor comprising PD0325901. In other embodiments, the mediacomprises a combination of inhibitors consisting of CHIR99021 andPD0325901. Either of these compounds can be obtained, for example, fromStemgent. In specific embodiments, CHIR99021 is present in the culturemedia at a concentration of about 0.5μ to about 3 μM, about 0.5μ toabout 3.5 μM, about 0.5 μM to about 4 μM, about 0.5 μM to about 1 μM,about 1 μM to about 1.5 μM, about 1.5 μM to about 2 μM, about 2 μM toabout 2.5 μM, about 2.5 to about 3 μM, 3 μM to about 3.5 μM. In furtherembodiments, CHIR99021 is present in the culture media at aconcentration of about 3 μM. In other embodiments, PD0325901 is presentin the culture media at a concentration of about 0.4 μM to about 1 uM,about 0.4 μM to about 1.5 uM, about 0.4 μM to about 2 μM, about 0.4 μMto about 0.8 μM, 0.8 μM to about 1.2 μM, about 1.2 to about 1.5 μM. Infurther embodiments, PD0325901 is present in the culture media at aconcentration of about 1 μM. In specific embodiments, CHIR99021 ispresent in the culture media at a concentration of about 3 μM andPD0325901 is present at a concentration of about 1 μM.

In one non-limiting embodiment, the culture media employed in thevarious methods and compositions disclosed herein is set forth in Table2. Within the context of this application, the media described in Table2 is referred to as 2i media.

TABLE 2 Non-limiting rat ES culture media. Reagent ConcentrationDMEM/F12 basal media 1x (50%) Neurobasal media 1x (50%)Penicillin/streptomycin 1% L-Glutamine  4 mM 2-Mercaptoethanol  0.1 mMN2 supplement 1x B27 supplement 1x LIF 100 U/ml PD0325901 (MEKinhibitor).  1 μM CHIR99021 (GSK inhibitor).  3 μM

Additional media that can be employed include those disclosed in Li etal. (2008) Cell 135:1299-1310, Yamamoto et al. (2012) Transgenic Rats21:743-755, Ueda et al. (2008) PLoS ONE 3(6):e2800, Meek et al. (2010)PLoS ONE 4 (12): e14225; Tong et al. (2010) Nature 467:211-213; USPatent Publication 2012/0142092, Buehr et al. (2008) Cell 135:1287-1298,Li et al. (135) Cell 1299-1310, each of which is herein incorporated byreference in their entirety. When employing such media, theconcentration and the source of LIF can be modified as outlined herein.In specific embodiments, the various culture media are used incombination with mouse LIF or an active variant or fragment thereof, andin even further embodiments, the various culture media comprise a mouseLIF or an active variant or fragment thereof at a concentration of about50 U/ml to about 100 U/ml, about 50 U/ml to about 150 U/ml, or about 100U/ml.

The temperature of the cultures of rat ES cells, both for the productionof the ES cell line and for the culturing and maintaining of the ES lineit typically carried out at about 35° C. to about 37.5° C. In specificembodiment, the temperature is 37.0° C. The culture is typically carriedout at 7.5% CO₂.

ii. Establishing a Rat ES Cell Line

Methods for generating a rat embryonic stem (ES) cell line are provided.Such methods comprises (a) culturing in vitro a first feeder cell layerand a morula-stage embryo, a blastocyst-stage embryo, or a rat embryo ata developmental stage between a morula-stage embryo and ablastocyst-stage embryo, wherein the zona pellucida of the rat embryohas been removed and wherein the culture conditions maintainpluripotency of the rat ES cell and comprises a media having mouseleukemia inhibitory factor (LIF) or an active variant or fragmentthereof; and, (b) transferring an outgrowth of an amorphousundifferentiated mass of rat ES cells to an in vitro culture wellcomprising a second feeder cell layer and culturing the outgrowth underconditions that maintain pluripotency of the rat ES cells and comprisesa media having mouse LIF or an active variant or fragment thereof, andthereby establishing a rat ES cell line. The various methods furthercomprise passaging and culturing in vitro the cells of the rat ES cellline, wherein each subsequent in vitro culturing comprises culturing therat ES cells on the feeder cell layer under conditions that maintainpluripotency of the rat ES cells and comprises a media having mouse LIFor an active variant or fragment thereof. Rat ES cell lines made by suchmethods are also provided.

Non-limiting examples of methods to establish a rat ES cell line havingthe various characteristics discussed herein are set forth in Example 3.Briefly, a rat embryo (i.e., a morula-stage embryo, a blastocyst-stageembryo, or a rat embryo at a developmental stage between a morula-stageembryo and a blastocyst-stage embryo) is flushed from the uteruses of afemale rat. In specific embodiments, a blastocyst or an 8 cell embryo isobtained. The zona pellucida is removed and the rat embryos are culturedon feeder cells (as discussed elsewhere herein) which, in specificembodiments, comprise a monolayer of mitotically inactivated mouseembryonic fibroblasts (MEFs). The cells of the morula-stage embryo, theblastocyst-stage embryo, or the rat embryo at a developmental stagebetween a morula-stage embryo and a blastocyst-stage embryo are culturedin vitro under conditions that maintain the ES rat cells and thereby aresufficient to maintain pluripotency and/or totipotency of the ES cells.Various media can be employed at this stage, including any of thevarious media discussed above which have LIF, including mouse LIF or anactive variant or fragment thereof, in the media.

The cultures are monitored for the presence of an outgrowth (anamorphous undifferentiated mass of cells). Once the outgrowth reaches anappropriate size, a given outgrowth is transferred to new feeder plateand cultured. Transfer is accompanied by enzymatic dissociation usingtrypsin in order to produce multiple colonies. This transfer is commonlyreferred to as “passage 1”. The speed at which each line expands varies.The media is changed as needed in order to maintain the pluripotency ortotipotency of the rat ES cells. The culture is monitored for thepresence of colonies having embryonic stem cell morphology. Suchmorphology includes one or more of the following characteristics: (a)round, circular mounds that rise above the monolayer of feeder cells;(b) cells that are packed tightly together such that cell boarders aredifficult to see; (c) smaller cell size; (d) small amount of cytoplasmand enlarged nucleus, (e) form sphere-like colonies when plated onfeeder cells in vitro. Once such colonies appear, the culturing cancontinue until reaching approximately 50% confluency. The colonies arethen transferred to a new feeder plate. Transfer is accompanied byenzymatic dissociation using trypsin in order to expand the number ofcolonies. This is referred to as “passage 2”. The cells are continued tobe cultured with feeder cells until they reach approximately 50%confluent, at which point the cells can undergo further passages tomaintain the cell lines or the lines can be frozen. See, also Tong etal. (2010) Nature 467 (9):211-215; Li et al. (2008) Cell 135:1299-1310,and Buehr et al. (2008) Cell 135:1287-1298, each of which is hereinincorporated by reference. Thus, in specific embodiments, the variousrat ES cells, cell lines and cell populations disclosed herein arecapable of being subcultured and maintaining the undifferentiated state.

In one non-limiting embodiment, the derivation of the rat ES cellsoccurs as follows. At day 0, female rats are euthanized and the oviductsand uterine horns are dissected out and place into a tissue culture dishcontaining warm N2B27 media. Media is flushed through the uterine hornsand oviducts to eject blastocysts into the media. The blastocysts arecollected and transfer to embryo culture dish containing KSOM+2i (1μMPD0325901, 3 μM CHIR99021). KSOM can be purchased from Millipore,catalog number is MR-106-D. The 2i media referred to herein comprisesthe media set forth in Table 2. The cells are culture overnight at 37°at 7.5% CO₂.

In other non-limiting embodiments, the rat ES cells are derived from8-cell embryos or frozen 8-cell embryos. The embryos are cultured in M2medium for 10 minutes at room temperature and are then transfer toKSOM+2i and culture overnight.

A non-limiting example for derivation of the rat ES cells is as follows:on Day 1, transferring cavitated embryos to 2i medium (Table 2) andculture overnight. The culturing is continued un-cavitated in KSOM+2i.On day 2, all remaining embryos are transferred to 2i medium, whether ornot they have cavitated. Culture continues overnight in 2i medium. Onday 3, embryos are incubated with acid tyrodes to remove the zonapellucida and washed 3 times in 2i medium to remove the acid tyrodes.Each embryo is deposited into a separate well feeder plate, in whicheach well contains a monolayer of mitotically inactivated mouseembryonic fibroblasts (MEFs). The cells are cultured overnight in 2imedium. On day 4 and 5, the cells plated embryos are monitored for thepresence of an outgrowth, an amorphous undifferentiated mass of cells.Outgrowths are ready for transfer when they are approximately twice thesize of the plated embryo. Each day, spent media is removed and replacedwith fresh 2i media. The outgrowths are transferred to new feeder wells,and again spent media is removed and the wells are washed with PBS. ThePBS is removed and trypsin is added and incubated for about 10 minutes.The trypsin reaction is stopped by the addition of 30 μl 2i media and10% FBS. The cells are gently dissociated and the entire content istransferred to a well in a feeder plate. This is referred to as Passage1 (P1). The cells are cultured overnight in 2i medium. On day 5-8,depending on how fast each line expands, the 2i media is changed eachday and culture is monitored for the presence of colonies with an ESCmorphology. Such ESC morphology is discussed in detail elsewhere herein.Culturing continues until colonies expand to about 50% confluency. Thecolonies are then trypsinzied and passaged as before into feeder wells.This is referred to as passage 2. Feeding and monitoring each line iscontinued until they are approximately 50% confluent. The cells aretrypsinized as usual. The trypsin is stopped with 2i media+10% FBS. Thecells are pelleted by centrifugation, and the cells in 400 μl FreezingMedium (70% 2i, 20% FBS, 10% DMSO). The cells can then be frozen. Thisis referred to passage 3.

iii. Maintaining and Passaging a Rat ES cell Line

Further provided are methods for maintaining or culturing a ratembryonic stem cell line. The method comprises culturing in vitro afeeder cell layer and a rat ES cell line, wherein the culture conditionsmaintain pluripotency of a rat embryonic stem (ES) cell and comprise amedia having mouse leukemia inhibitory factor (LIF) or an active variantor fragment thereof. Such methods employ the culture media and feedercell layer as outlined above. In one embodiment, the rat ES cell linecan be passaged at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50 or more times. In specificembodiments, the rat ES cells can be passaged up to at least 11 times ina medium comprising a GSK3 inhibitor and a MEK inhibitor withoutdecreasing its targeting efficiency or germline transmission efficiencyof a targeted genetic modification.

The rat ES cell lines are fed and monitored. In specific embodiments,passage occurs when the culture is approximately 30%, 40%, 50%, or 60%confluent. In other embodiments, passage occurs when the culture is 50%confluent. Depending on how fast each line expands, the passages canoccur every 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 55, 60, 65, 70, 75,80, 90, 95 or 100 hours. In other embodiments, the time between passagesranges between 24 hours and 96 hours, between about 30 and 50 hours,between about 25 and 75 hours, between about 50 and 96 hours, betweenabout 25 and 75 hours, between about 35 and 85 hours, or between about40 and 70 hours. In one embodiment, the rat ES cell, cell line or cellpopulation as disclosed herein has a doubling time ranging from about 24hours to about 36 hours. In one embodiment, the rat ES cell has adoubling time of 25 hours.

The various rat ES cell lines when derived and maintained as outlinedherein can have one or more any the following properties:

(a) have germ-line competency, meaning when the rat ES cell is implantedinto a rat host embryo, the genome of the rat ES cell line istransmitted into an offspring;

(b) have germ-line competency following targeted genetic modification,meaning when the rat ES cell is implanted into a rat host embryo, thetargeted genetic modification within the genome of the rat ES cell line,is transmitted into an offspring;

(c) have pluripotency in vitro;

(d) have totipotency in vitro;

(e) when cultured in vitro loosely adhere to a feeder cell layer;

(f) when cultured in vitro form sphere-like colonies when plated on afeeder cell layer in vitro;

(g) maintain pluripotency when cultured in vitro under conditionscomprising a feeder cell layer that is not genetically modified toexpress leukemia inhibitory factor (LIF), wherein the culture mediacomprises a sufficient concentration of LIF;

(h) maintain pluripotency when cultured in vitro under conditionscomprising a feeder cell layer, wherein the culture media comprisesmouse LIF or an active variant or fragment thereof;

(i) comprise a molecular signature characterized by

-   -   i) the expression of one or more of rat ES cell-specific genes        comprising Adherens Junctions Associate Protein 1 (Ajap1),        Claudin 5 (Cldn5), Cdc42 guanine nucleotide exchange factor 9        (Arhgef9), Calcium/calmodulin-dependent protein kinase IV        (Camk4), ephrin-A1 (Efna1), EPH receptor A4 (Epha4), gap        junction protein beta 5 (Gjb5), Insulin-like growth factor        binding protein-like 1 (Igfbpl1), Interleukin 36 beta (Il1f8),        Interleukin 28 receptor, alpha (Il28ra), left-right        determination factor 1 (Lefty1), Leukemia inhibitory factor        receptor alpha (Lifr), Lysophosphatidic acid receptor 2 (Lpar2),        Neuronal pentraxin receptor (Ntm), Protein tyrosine phosphatase        non-receptor type 18 (Ptpn18), Caudal type homeobox 2 (Cdx2),        Fibronectin type III and ankyrin repeat domains 1 (Fank1),        Forkhead box E1 (thyroid transcription factor 2) (Foxe1),        Hairy/enhancer-of-split related with YRPW motif 2 (Hey2),        Forkhead box E1 (thyroid transcription factor 2) (Foxe1),        Hairy/enhancer-of-split related with YRPW motif 2 (Hey2),        Lymphoid enhancer-binding factor 1 (Lef1), Sal-like 3        (Drosophila) (Sall3), SATB homeobox 1 (Satb1), miR-632, or a        combination thereof;    -   ii) the expression of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more        of the rat ES cell-specific genes comprising Adherens Junctions        Associate Protein 1 (Ajap1), Claudin 5 (Cldn5), Cdc42 guanine        nucleotide exchange factor 9 (Arhgef9),        Calcium/calmodulin-dependent protein kinase IV (Camk4),        ephrin-A1 (Efna1), EPH receptor A4 (Epha4), gap junction protein        beta 5 (Gjb5), Insulin-like growth factor binding protein-like 1        (Igfbpl1), Interleukin 36 beta (Il1f8), Interleukin 28 receptor,        alpha (Il28ra), left-right determination factor 1 (Lefty1),        Leukemia inhibitory factor receptor alpha (Lifr),        Lysophosphatidic acid receptor 2 (Lpar2), Neuronal pentraxin        receptor (Ntm), Protein tyrosine phosphatase non-receptor type        18 (Ptpn18), Caudal type homeobox 2 (Cdx2), Fibronectin type III        and ankyrin repeat domains 1 (Fank1), Forkhead box E1 (thyroid        transcription factor 2) (Foxe1), Hairy/enhancer-of-split related        with YRPW motif 2 (Hey2), Forkhead box E1 (thyroid transcription        factor 2) (Foxe1), Hairy/enhancer-of-split related with YRPW        motif 2 (Hey2), Lymphoid enhancer-binding factor 1 (Lef1),        Sal-like 3 (Drosophila) (Sall3), SATB homeobox 1 (Satb1),        miR-632, or a combination thereof;    -   iii) at least a 20-fold increase in the expression of one or        more of the rat ES cell-specific genes as set forth in Table 14        when compared to a F1H4 mouse ES cell;    -   iv) at least a 20-fold increase in the expression of at least 2,        3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25 or more of the rat ES cell-specific genes as        set forth in Table 14 when compared to a F1H4 mouse ES cell;    -   v) the expression of one or more of rat ES cell-specific genes        as set forth in Table 13;    -   vi) the expression of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,        40, 45, 50 or more of the rat ES cell-specific genes as set        forth in Table 13;    -   vii) at least a 20-fold increase in the expression of one or        more of the rat ES cell-specific genes as set forth in Table 13        when compared to a F1H4 mouse ES cell;    -   viii) at least a 20-fold increase in the expression of at least        2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,        20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more of the rat ES        cell-specific genes as set forth in Table 13 when compared to a        F1H4 mouse ES cell;    -   ix) at least a 20-fold decrease in the expression of one or more        of the rat ES cell-specific genes as set forth in Table 12 when        compared to a F1H4 mouse ES cell; and/or    -   x) at least a 20-fold decrease in the expression of at least 2,        3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more of the rat ES        cell-specific genes as set forth in Table 12 when compared to a        F1H4 mouse ES cell;    -   xi) any combination of expression of the rat ES cell-specific        genes of parts (i)-xii) a relative expression level of        pluripotency markers as shown in Table 15 for at least 2, 3, 4,        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 of the        listed pluripotency markers. See, pluripotency ranking column of        Table 15 for relative expression levels;    -   xiii) a relative expression level of the mesodermal markers as        shown in Table 15 for at least 2, 3, or 4 of the listed        mesodermal markers. See, mesodermal ranking column in Table 15        for relative expression levels;    -   xiv) a relative expression level of endodermal markers as shown        in Table 15 for at least 2, 3, 4, 5, or 6 of the listed        endodermal markers. See, endodermal ranking column in Table 15        for relative expression levels;    -   xv) a relative expression level of neural markers as shown in        Table 15 for at least 2 and 3 of the listed neural markers. See,        neural ranking column in table 15 for relative expression        levels;    -   xvi) a relative expression level of trophectoderm markers as        shown in Table 15 for the listed trophectoderm markers. See,        trophectoderm ranking column in table 15 for relative expression        levels;    -   xvii) any relative expression level of one or more (2, 3, 4, 5,        6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,        23, 24, 25, 26, 27, 28, 29 or 30) of the pluripotency markers,        mesodermal markers, endodermal markers, neural markers and/or        trophectoderm markers set forth in Table 15;    -   xviii) the relative expression level of each of the markers set        forth in table 15;    -   xix) any combination of the signatures set forth in xii-xiix;        and/or    -   xx) any combination of the signature set forth in i-xiix; have        the ability to produce a F0 rat;

(k) capable of being subcultured and maintaining the undifferentiatedstate;

(l) have the same number of chromosomes a normal rat cell; and/or

(m) maintain pluripotency in vitro without requiring paracrine LIFsignaling.

(n) have self renewal, meaning they divide indefinitely whilemaintaining pluripotency.

Such properties of a given rat ES cell line can be present at any one ofthe passage, including at passages 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50 or later.

Thus, in one non-limiting embodiment, an in vitro culture comprising afeeder cell layer and a population of rat ES cells is provided, whereinthe in vitro culture conditions maintain pluripotency of the rat EScells and comprises a media having mouse leukemia inhibitory factor(LIF) or an active variant or fragment thereof. In specific embodiments,the rat ES cells cultured under such conditions maintains pluripotencyof at least 50% of cell population over a period of at least 10passages, maintains pluripotency of at least 60% of cell population overa period of at least 10 passages, maintains pluripotency of at least 70%of cell population over a period of at least 10 passages, maintainspluripotency of at least 75% of cell population over a period of atleast 10 passages, maintains pluripotency of at least 80% of cellpopulation over a period of at least 10 passages, maintains pluripotencyof at least 85% of cell population over a period of at least 10passages, maintains pluripotency of at least 90% of cell population overa period of at least 10 passages, or maintains pluripotency of at least95% of cell population over a period of at least 10 passages.

Further provided herein are in vitro cultures comprising the various ratES cells, cell populations and cell lines disclosure herein, as well as,culturing kits for these various ES cells. For example, as discussedabove, in specific embodiments, the various rat ES cells provided hereinhave a one or more of the following characteristics: (1) when culturedin vitro loosely adhere to a feeder cell layer; (2) when cultured invitro they form sphere-like colonies when plated on a feeder cell layerin vitro; (3) they maintain pluipotency when cultured in vitro underconditions comprising a feeder cell layer that is not geneticallymodified to express leukemia inhibitory factor (LIF), wherein theculture media comprises a sufficient concentration of LIF; and/or (4)they capable of being subcultured and maintaining the undifferentiatedstate. Moreover, the rat ES cell populations of any of these in vitrocultures can comprise, for example, a population of cells in which atleast 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% of the cells within the population are euploid, are diploid and/orhave 42 chromosomes.

One method of culturing a rat embryonic stem cells in vitro comprises(a) providing an in vitro culture comprising a feeder cell layer and apopulation of isolated rat embryonic stem (ES) cells; and (b) culturingthe in vitro culture under conditions which are sufficient to maintainpluipotency or totipotency of the isolated rat embryonic stem (ES) cell,and wherein the rat ES cells form colonies that loosely adhere to thefeeder cell layer. “Loose adherence” or “adhere loosely” means that, ifthe culture dish is undisturbed for a period of time (minimum 8 hours),some colonies will adhere to the feeders such that they can maintainadherence if the dish is gently moved. In smaller wells (where the mediamoves less), loose adherence can happen faster. In either case, thesecolonies can be dislodged by either a) swirling the media in the dish orby gently pipetting media across the surface of the feeders. Themorphology of these loosely adherent colonies is still spherical. Insuch instances the rat ES cells form sphere-like colonies when plated ona feeder cell layer in vitro. Such sphere-like colonies are shown forexample in FIG. 1.

iv. Kits and In Vitro Cultures

The rat ES cells and rat ES cell lines provided herein can be containedwithin a kit or an article of manufacture. In specific embodiments, thekit or article of manufacture comprises any of the rat ES cell lines orpopulations disclosed herein. The kit can further comprise any culturemedia that maintains the rat ES cell, including media that maintains thepluripotency of the rat ES cells. Such media can comprise culture mediahaving mouse LIF or an active variant or fragment thereof, as discussedin greater detail elsewhere herein. The media within the kit can furthercomprise a MEK inhibitor and a GSK-3 inhibitor, or alternatively, themedia within the kit can further comprise a combination of inhibitorsconsisting of a MEK inhibitor and a GSK-3 inhibitor. In specificembodiments, the media in the kit comprises a MEK inhibitor comprisingPD0325901 and/or a GSK-3 inhibitor comprising CHIR99021. Any of thevarious media discussed herein, can be contained within the kit.

Further provided is a kit or article of manufacture that comprises anyof the rat ES cell lines or populations disclosed herein, any of thevarious media disclosed herein, and a population of feeder cells. In oneembodiment, the feeder cells in the kit or article of manufacture arenot genetically modified to express LIF and/or the feeder cellscomprises mitotically inactivated mouse embryonic fibroblasts (MEFs).Any of the other feeder cells disclosed herein can be employed in thekit or article of manufacture.

IV. Genetic Modification of Rat Embryonic Stem (ES) Cells

The various rat ES cells and cell lines disclosed herein can be modifiedto contain at least one targeted genetic modification. Thus, variousmethods are provided for genetically modifying an isolated rat embryonicstem (ES) cell as disclosed herein. The method comprises introducinginto the genome of an isolated rat ES cell disclosed herein a targetedgenetic modification to form a genetically modified rat ES cell. Thetargeted genetic modification can comprise any modification to thegenome of rat ES including, for example, an insertion, a deletion, aknockout, a knockin, a mutation, or a combination thereof. In oneembodiment, the targeted genetic modification comprises insertion of aheterologous polynucleotide into the genome of the rat ES cell. As usedherein, “heterologous” in reference to a sequence is a sequence thatoriginates from a foreign species, or, if from the same species, issubstantially modified from its native form in composition and/orgenomic locus by deliberate human intervention.

In one aspect, an isolated rat ES cell or rat ES cell line is providedthat is capable of sustaining pluripotency following one or more geneticmodifications in vitro and that is capable of transmitting a geneticallymodified genome to a germline of an F1 generation. Thus, the rat ES cellmaintains its pluripotency to develop into a plurality of cell typesfollowing the one or more serial genetic modifications in vitro (e.g.,two, three, four, five, or six or more serial genetic modifications). Inother embodiments, multiple targeted genetic modifications are made in agiven rat ES cell, including, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13 14, 15 or more. As such, multiple heterologouspolynucleotides can also be integrated into the genome, including forexample, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 14, 15 or more.

In one embodiment, following any one of the one to 15 serial geneticmodifications, the genetically modified rat ES cells upon exposure todifferentiation medium are capable of differentiation into a pluralityof cell types. In one embodiment, following any one of the one to 15serial genetic modifications, the genetically modified rat ES cells arecapable of being maintained in an undifferentiated state in culture. Inone embodiment, the genetically modified and cultured rat ES cells inthe undifferentiated state, when employed as donor cells in a rat hostembryo, populate the embryo and form a blastocyst comprising the one tofifteen genetic modifications. In one embodiment, the blastocyst, whenimplanted into a surrogate mother under conditions suitable forgestation, develops into an F0 rat progeny that comprises the one to 15genetic modifications.

Various methods for making targeted genetic modifications within agenome of a rat ES cell can be used. For example, in one instance, thetargeted genetic modification employs a system that will generate atargeted genetic modification via a homologous recombination event. Inother instances, the rat ES cells can be modified using nuclease agentsthat generate a single or double strand break at a targeted genomiclocation. The single or double-strand break is then repaired by thenon-homologous end joining pathway (NHEJ). Such systems find use, forexample, in generating targeted loss of function genetic modifications.See, for example, Tesson et al. (2011) Nature Biotechnology 29:695-696,herein incorporated by reference. Such agents include, TranscriptionActivator-Like Effector Nuclease (TALEN) (WO 2010/079430; Morbitzer etal. (2010) PNAS 10.1073/pnas.1013133107; Scholze & Boch (2010) Virulence1:428-432; Christian et al. Genetics (2010) 186:757-761; Li et al.(2010) Nuc. Acids Res. (2010) doi:10.1093/nar/gkq704; and Miller et al.(2011) Nature Biotechnology 29:143-148; US Patent Application No.2011/0239315 A1, 2011/0269234 A1, 2011/0145940 A1, 2003/0232410 A1,2005/0208489 A1, 2005/0026157 A1, 2005/0064474 A1, 2006/0188987 A1, and2006/0063231 A1 (each hereby incorporated by reference); a zinc-fingernuclease (ZFN) (US20060246567; US20080182332; US20020081614;US20030021776; WO/2002/057308A2; US20130123484; US20100291048; and,WO/2011/017293A2, each of which is herein incorporated by reference); ameganuclease (see, Epinat et al., (2003) Nucleic Acids Res 31:2952-62;Chevalier et al., (2002) Mol Cell 10:895-905; Gimble et al., (2003) MolBiol 334:993-1008; Seligman et al., (2002) Nucleic Acids Res 30:3870-9;Sussman et al., (2004) J Mot Biol 342:31-41; Rosen et al., (2006)Nucleic Acids Res 34:4791-800; Chames et al., (2005) Nucleic Acids Res33:e178; Smith et al., (2006) Nucleic Acids Res 34:e149; Gruen et al.,(2002) Nucleic Acids Res 30:e29; Chen and Zhao, (2005) Nucleic Acids Res33:e154; WO2005105989; WO2003078619; WO2006097854; WO2006097853;WO2006097784; and WO2004031346); and a CRISPR/Cas system (Mali P et al.(2013) Science 2013 Feb. 15; 339(6121):823-6; Jinek M et al. Science2012 Aug. 17; 337(6096):816-21; Hwang W Y et al. Nat Biotechnol 2013March; 31(3):227-9; Jiang W et al. Nat Biotechnol 2013 March;31(3):233-9; and, Cong L et al. Science 2013 Feb. 15; 339(6121):819-23,each of which is herein incorporated by reference).

In other embodiments, the targeted genomic modification can be made byemploying a homologous recombination targeting vector. In suchinstances, the targeting vector comprises the insert polynucleotide andfurther comprises an upstream and a downstream homology arm which flankthe insert polynucleotide. The homology arms which flank the insertpolynucleotide correspond to genomic regions within the targeted genomiclocus. For ease of reference, the corresponding genomic regions withinthe targeted genomic locus are referred to herein as “target sites”.Thus, in one example, a targeting vector can comprise a first insertpolynucleotide flanked by a first and a second homology armcorresponding to a first and a second target site located at thetargeted genomic locus. The targeting vector thereby aids in theintegration of the insert polynucleotide into the targeted genomic locusthrough a homologous recombination event that occurs between thehomology arms and the corresponding target sites within the genome ofthe cell.

As used herein, a homology arm and a target site “correspond” or are“corresponding” to one another when the two regions share a sufficientlevel of sequence identity to one another to act as substrates for ahomologous recombination reaction. By “homology” is meant DNA sequencesthat are either identical or share sequence identity to a correspondingsequence. The sequence identity between a given target site and thecorresponding homology arm found on the targeting vector can be anydegree of sequence identity that allows for homologous recombination tooccur. For example, the amount of sequence identity shared by thehomology arm of the targeting vector (or a fragment thereof) and thetarget site (or a fragment thereof) can be at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% sequenceidentity, such that the sequences undergo homologous recombination.Moreover, a corresponding region of homology between the homology armand the corresponding target site can be of any length that issufficient to promote homologous recombination at the cleavedrecognition site.

In specific embodiments, the isolated rat ES cell, cell line or cellpopulation exhibits a homologous recombination efficiency of at least2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, atleast 8%, at least 9%, at least 10%, at least 11%, at least 12%, atleast 13%, at least 14%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, or atleast 80%. In one embodiment, the homologous recombination efficiencyemploying the rat ES cell is greater than 4%.

In specific embodiments, a selection marker is employed when generatinga targeted genetic modification in a rat ES cell. The polynucleotideencoding the selection marker can be present on the targeting vectorwhich is designed to introduce the targeted genetic modification intothe genome, or it can be found on a separate plasmid or vector. Thepolynucleotide encoding the selection marker can be contained within inexpression cassette. The various components of such expression cassettesare discussed in further detail elsewhere herein. Various selectionmarkers can be used in the methods and compositions disclosed herein.Such selection markers can, for example, impart resistance to anantibiotic such as G418, hygromycin, blastocidin, puromycin or neomycin.Such selection markers include neomycin phosphotransferase (neo^(r)),hygromycin B phosphotransferase (hyg^(r)), puromycin N-acetyltransferaseand blasticidin S deaminase (bsr^(r)). In still other embodiments, theselection marker is operably linked to an inducible promoter and theexpression of the selection marker is toxic to the cell. Non-limitingexamples of such selection markers include xanthine/guaninephosphoribosyl transferase (gpt), hahypoxanthine-guaninephosphoribosyltransferase (HGPRT) or herpes simplex virus thymidinekinase (HSV-TK). See, for example, Santerre et al. (1984) Gene30:147-56; Joyner (1999) The Practical Approach Series, 293; Santerre etal. (1984) Gene 30:147-56; Bernard et al. (1985) Exp Cell Res158:237-43; Giordano and McAllister (1990) Gene, 88:285-8; Izumi et al.(1991) Exp Cell Res 197:229-33), each of which is herein incorporated byreference in their entirety. In specific embodiments, the neoRselectable marker is the neomycin phosphotransferase (neo) gene of Becket al. (1982) Gene, 19:327-36, which is herein incorporated byreference. The neoR selection maker is that used in U.S. Pat. Nos.7,205,148 or 6,596,541, each of which are herein incorporated byreference.

In specific embodiments, the selection marker employed is anon-attenuated selection marker. A “non-attenuated selection marker”comprises a selection marker that retains the activity of the nativepolypeptide or the selection marker has an increased activity whencompared to the native form of the polypeptide. An increased in activityof a selection marker can comprise any statistically significantincrease in activity including, for example, an increase of at least 5%,10%, 20%, 30%, 40%, 50%, 60%, 70% 80% or higher. Thus, a non-attenuatedselection marker when expressed in a host cell will allow for a higherpercentage of the host cells to survive in the presence of a higherconcentration of the selection agent than when employing an attenuatedselection marker. Non-attenuated selection markers include, for example,a neomycin non-attenuate selection marker. See, for example, Beck et al.(1982) Gene, 19:327-36 or in U.S. Pat. Nos. 7,205,148 or 6,596,541, eachof which are herein incorporated by reference.

In other instances, the increased activity of a selection marker whencompared to an attenuated selection marker and/or the wild type (native)selection marker can result from increasing the copy number of eitherthe non-attenuated, attenuated or native selection marker within thegenome of the rat ES cell. As such, a given rat ES cell can comprisewithin its genome at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copiesof a given selection marker (i.e., a non-attenuated selection marker, anattenuated selection marker or a native (wildtype) selection marker).

The various selection markers employed are encoded by a polynucleotideoperably linked to a promoter active in the rat ES cell. In specificembodiments, the increase in the activity of the selection marker canresult from an increase in expression of the selection marker. Thus, apromoter can be employed to elevate the expression levels of a givenselection marker. Promoters of interest include, but are not limited to,the CMV promoter, the PGK promoter and the CAG promoter. In oneembodiment, the human Ubiquitin (hUb) promoter is used to express theselection marker. See, Valenzuela et al. (2003) Nature Biotechnology21:652-659, herein incorporate by reference in its entirety.

In one embodiment, the rat ES cell maintains its pluripotency to developinto a plurality of cell types following a single round ofelectroporation with an exogenous nucleic acid. In another embodiment,the rat ES cell maintains its pluripotency to develop into a pluralityof cell types following a second round of electroporation with anexogenous nucleic acid, following a third round of electroporation withan exogenous nucleic acid, following a fourth round of electroporationwith an exogenous nucleic acid, following a fifth round ofelectroporation with an exogenous nucleic acid, following a sixth roundof electroporation with an exogenous nucleic acid, following a seventhround of electroporation with an exogenous nucleic acid, following aneighth round of electroporation with an exogenous nucleic acid,following a ninth round of electroporation with an exogenous nucleicacid, following a tenth round of electroporation with an exogenousnucleic acid, following an eleventh round of electroporation with anexogenous nucleic acid, following a twelfth round of electroporationwith an exogenous nucleic acid, following a thirteenth round ofelectroporation with an exogenous nucleic acid, following a fourteenthround of electroporation with an exogenous nucleic acid and/or followinga fifteenth round of electroporation with an exogenous nucleic acid. Inother embodiments, the rat ES cell is capable of transmitting a targetedgenetic modification into progeny following a successive round ofelectroporation (i.e., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or morerounds of electroporation).

i. Introducing Sequences into a Rat Embryonic Stem Cell

The methods provided herein comprise introducing into a cell with one ormore polynucleotides or polypeptide constructs comprising the variouscomponents needed to make the targeted genomic modification.“Introducing” means presenting to the cell the sequence (polypeptide orpolynucleotide) in such a manner that the sequence gains access to theinterior of the cell. The methods provided herein do not depend on aparticular method for introducing any component of the targeted genomicintegration system into the cell, only that the polynucleotide gainsaccess to the interior of a least one cell. Methods for introducingpolynucleotides into various cell types are known in the art andinclude, but are not limited to, stable transfection methods, transienttransfection methods, and virus-mediated methods. Such method include,but are not limited to, electroporation, intracytoplasmic injection,viral infection (including adenovirus, lentivirus, and retrovirusvectors), transfection, lipid-mediated transfection and/orNucleofaction™. See, for example, Stadtfeld et al. (2009) Nature Methods6(5):329-330; Yusa et al. (2009) Nat. Methods 6:363-369; Woltj en et al.(2009) Nature 458, 766-770. Such methods include, but are not limitedto, direct delivery of DNA such as by ex vivo transfection (Wilson etal., Science, 244:1344-1346, 1989, Nabel and Baltimore, Nature326:711-713, 1987), optionally with Fugene6 (Roche) or Lipofectamine(Invitrogen), by injection (U.S. Pat. Nos. 5,994,624, 5,981,274,5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and5,580,859, each incorporated herein by reference), includingmicroinjection (Harland and Weintraub, J. Cell Biol., 101:1094-1099,1985; U.S. Pat. No. 5,789,215, incorporated herein by reference); byelectroporation (U.S. Pat. No. 5,384,253, incorporated herein byreference; Tur-Kaspa et al., Mol. Cell Biol., 6:716-718, 1986; Potter etal., Proc. Nat'l Acad. Sci. USA, 81:7161-7165, 1984); by calciumphosphate precipitation (Graham and Van Der Eb, Virology, 52:456-467,1973; Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752, 1987; Rippe etal., Mol. Cell Biol., 10:689-695, 1990); by using DEAE-dextran followedby polyethylene glycol (Gopal, Mol. Cell Biol., 5:1188-1190, 1985); bydirect sonic loading (Fechheimer et al., Proc. Nat'l Acad. Sci. USA,84:8463-8467, 1987); by liposome mediated transfection (Nicolau andSene, Biochim. Biophys. Acta, 721:185-190, 1982; Fraley et al., Proc.Nat'l Acad. Sci. USA, 76:3348-3352, 1979; Nicolau et al., MethodsEnzymol., 149:157-176, 1987; Wong et al., Gene, 10:87-94, 1980; Kanedaet al., Science, 243:375-378, 1989; Kato et al., Biol. Chem.,266:3361-3364, 1991) and receptor-mediated transfection (Wu and Wu,Biochemistry, 27:887-892, 1988; Wu and Wu, J. Biol. Chem.,262:4429-4432, 1987); and any combination of such methods, each of whichis incorporated herein by reference.

In some embodiments, the cells employed in the methods and compositionshave a DNA construct stably incorporated into their genome. “Stablyincorporated” or “stably introduced” means the introduction of apolynucleotide into the cell such that the nucleotide sequenceintegrates into the genome of the cell and is capable of being inheritedby progeny thereof. Any protocol may be used for the stableincorporation of the DNA constructs or the various components employedto generate the targeted genomic modification.

Transfection protocols as well as protocols for introducing polypeptidesor polynucleotide sequences into cells may vary. Non-limitingtransfection methods include chemical-based transfection methods includethe use of liposomes; nanoparticles; calcium phosphate (Graham et al.(1973). Virology 52 (2): 456-67, Bacchetti et al. (1977) Proc Natl AcadSci USA 74 (4): 1590-4 and, Kriegler, M (1991). Transfer and Expression:A Laboratory Manual. New York: W. H. Freeman and Company. pp. 96-97);dendrimers; or cationic polymers such as DEAE-dextran orpolyethylenimine. Non chemical methods include electroporation;Sono-poration; and optical transfection. Particle-based transfectioninclude the use of a gene gun, magnet assisted transfection (Bertram, J.(2006) Current Pharmaceutical Biotechnology 7, 277-28). Viral methodscan also be used for transfection.

A non-limiting example, of a method of introduction a heterologouspolynucleotide into a rat ES cell follows. The rat ES cells, asdescribed herein, are passaged for about 24 to about 48 prior toelectroporation. About 24 hours prior to electroporation the media ischanged to RVG2i+ROCKi (10 μM Y-27632). The rat ES cells are trypsinizedand the rat ES cells are isolated. The rat ES cells are suspended toachieve a final cell concentration of about 2×10{circumflex over ( )}6to about 10×10{circumflex over ( )}6 cells per 75 ul. About 75λ, of ratES cells are added to about 50λ DNA comprising the heterologouspolynucleotide and about 125λ EP buffer is added. In one non-limitingembodiment, the electroporation is carried out with the followingparameters: 400V; 400V; Ω; 100 μF. The cells are then cultured in RVG2iand 10 μM ROCKi and can be transferred onto feeder cells.

ii. Selecting Rat Embryonic Stem Cells Having a Targeted GenomicModification

Various method are provided for selecting and maintaining rat ES cellshaving stably incorporated into their genome a targeted geneticmodification. In one non-limiting example, when introducing aheterologous polynucleotide into a rat ES cell, the method can comprise(a) providing an in vitro population of rat ES cells; (b) introducinginto at least one rat ES cell a heterologous polynucleotide comprising aselection marker operably linked to a promoter active the rat ES cell;and, (c) culturing in vitro the rat ES cell population in an alternatingfirst and second culture media wherein the first culture media comprisesan effective amount of a selection agent for a first time period and thesecond culture media do not comprise the selection agent, wherein the invitro culture conditions maintain pluripotency or totipotency; andthereby selecting the rat ES cell having stably integrated into itsgenome the heterologous polynucleotide. The various methods by which therat ES cell having the targeted genetic modification can be selected ina given population can employ an in vitro culture system which allowsthe rat ES cells to maintain pluripotency. Thus, any of the in vitroculture media and feeder cells discussed herein can be employed.

In specific embodiments, the first and the second culture media arealternated about every 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40 hours or more. In a specific embodiment, the first and secondculture media are alternated every 24 hours.

Any appropriate selection marker can be used and the correspondingselection agent will be present at an effective concentration with theculture media. Such selection markers include any of the native,attenuated or non-attenuated selection marker discussed herein. In oneembodiment, the selection marker employed imparts resistance to anantibiotic, including for example, G418. Non-limiting selection markerscomprise neomycin phosphotransferase II (nptII) or hygromycinphosphotransferase (hpt).

The concentration of the selection agent is such as to allow for theselection of a rat ES cell having the selection marker while maintainingthe pluipotency of the rat ES cells that are present within the culture.When employing, for example, G418, the concentration of the G418 in theselection media can range from about 50 ug/ml to about 125 ug/ml, about60 ug/ml to about 125 ug/ml, about 70 ug/ml to about 125 ug/ml, about 80ug/ml to about 125 ug/ml, about 90 ug/ml to about 125 ug/ml, about 100ug/ml to about 125 ug/ml, about 110 ug/ml to about 125 ug/ml, about 80ug/ml to about 100 ug/ml, about 65 ug/ml to about 85 ug/ml, about 70ug/ml to about 80 ug/ml. In one embodiment, the concentration of G418 inthe culture is 75 μg/ml.

The media employed in the selection allows the rat embryonic stem cellsto retain pluripotency. Such media is described in detail elsewhereherein.

The selection protocol can be initiated at any time following theintroduction of the polynucleotide encoding the selection marker intothe genome of the rat ES cell. In specific embodiments, the selectionprotocol begins 10, 15, 20, 24, 30, 35, 40, 50, 60 or more hours afterthe introduction of the selection marker into the rat ES cell. In oneembodiment, the selection protocol beings about 2 days following theintroduction of the polynucleotide encoding the selection marker.

A non-limiting selection protocol employing G418 is as follows. Day 2,(2^(nd) day after the introduction of the polynucleotide encoding theselection marker) the population of rat ES cells is incubated cells in2i media and G418 at 75 μg/ml. At day 3, the population of rat ES cellsare incubated cells in 2i media without G418. At day 4, the populationof rat ES cells are incubated in 2i media and G418 at 75 μg/ml. At day5, the population of rat ES cells are incubated cells in 2i mediawithout G418. At day 6, the population of rat ES cells are incubated in2i media and G418 at 75 μg/ml. At day 7, the population of rat ES cellsis incubated in 2i media without G418. At day 8, the population of ratES cells is incubated in in 2i media without G418 and 75 μg/ml. At day9, the population of rat ES cells is incubated cells in 2i media withoutG418. At day 10, the population of rat ES cells is incubated cells in 2imedia and G418 at 75 μg/ml. At day 11, the population of rat ES cells isincubated cells in 2i media without G418. At day 12, colonies are pickedfor expansion and screening.

Following the selection of the rat ES cells having the selection marker,the colonies can be expanded. In specific embodiments, the period forexpansion can be about 1, 2, 3, 4, 5, or more days in a culturecondition that maintains the pluripotency of the cells. In onenon-limiting embodiment, the selected colonies are expanded for 3 days.In a further embodiment, the media employed is a 2i media. Each clonecan then be passed and further expanded.

The rat ES cells and cell lines having one or more of the targetedgenetic modifications can have one or more any the following properties:

(a) have germ-line competency following targeted genetic modification,meaning when the rat ES cell is implanted into a rat host embryo, thetargeted genetic modification within the genome of the rat ES cell line,is transmitted into an offspring;

(b) have pluripotency in vitro;

(c) have totipotency in vitro;

(d) when cultured in vitro loosely adhere to a feeder cell layer;

(e) when cultured in vitro form sphere-like colonies when plated on afeeder cell layer in vitro;

(f) maintain pluripotency when cultured in vitro under conditionscomprising a feeder cell layer that is not genetically modified toexpress leukemia inhibitory factor (LIF), wherein the culture mediacomprises a sufficient concentration of LIF;

(g) maintain pluripotency when cultured in vitro under conditionscomprising a feeder cell layer, wherein the culture media comprisesmouse LIF or an active variant or fragment thereof;

(h) comprise a molecular signature characterized by

-   -   i) the expression of one or more of rat ES cell-specific genes        comprising Adherens Junctions Associate Protein 1 (Ajap1),        Claudin 5 (Cldn5), Cdc42 guanine nucleotide exchange factor 9        (Arhgef9), Calcium/calmodulin-dependent protein kinase IV        (Camk4), ephrin-A1 (Efna1), EPH receptor A4 (Epha4), gap        junction protein beta 5 (Gjb5), Insulin-like growth factor        binding protein-like 1 (Igfbpl1), Interleukin 36 beta (Il1f8),        Interleukin 28 receptor, alpha (Il28ra), left-right        determination factor 1 (Lefty1), Leukemia inhibitory factor        receptor alpha (Lifr), Lysophosphatidic acid receptor 2 (Lpar2),        Neuronal pentraxin receptor (Ntm), Protein tyrosine phosphatase        non-receptor type 18 (Ptpn18), Caudal type homeobox 2 (Cdx2),        Fibronectin type III and ankyrin repeat domains 1 (Fank1),        Forkhead box E1 (thyroid transcription factor 2) (Foxe1),        Hairy/enhancer-of-split related with YRPW motif 2 (Hey2),        Forkhead box E1 (thyroid transcription factor 2) (Foxe1),        Hairy/enhancer-of-split related with YRPW motif 2 (Hey2),        Lymphoid enhancer-binding factor 1 (Lef1), Sal-like 3        (Drosophila) (Sall3), SATB homeobox 1 (Satb1), miR-632, or a        combination thereof;    -   ii) the expression of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more        of the rat ES cell-specific genes comprising Adherens Junctions        Associate Protein 1 (Ajap1), Claudin 5 (Cldn5), Cdc42 guanine        nucleotide exchange factor 9 (Arhgef9),        Calcium/calmodulin-dependent protein kinase IV (Camk4),        ephrin-A1 (Efna1), EPH receptor A4 (Epha4), gap junction protein        beta 5 (Gjb5), Insulin-like growth factor binding protein-like 1        (Igfbpl1), Interleukin 36 beta (Il1f8), Interleukin 28 receptor,        alpha (Il28ra), left-right determination factor 1 (Lefty1),        Leukemia inhibitory factor receptor alpha (Lifr),        Lysophosphatidic acid receptor 2 (Lpar2), Neuronal pentraxin        receptor (Ntm), Protein tyrosine phosphatase non-receptor type        18 (Ptpn18), Caudal type homeobox 2 (Cdx2), Fibronectin type III        and ankyrin repeat domains 1 (Fank1), Forkhead box E1 (thyroid        transcription factor 2) (Foxe1), Hairy/enhancer-of-split related        with YRPW motif 2 (Hey2), Forkhead box E1 (thyroid transcription        factor 2) (Foxe1), Hairy/enhancer-of-split related with YRPW        motif 2 (Hey2), Lymphoid enhancer-binding factor 1 (Lef1),        Sal-like 3 (Drosophila) (Sall3), SATB homeobox 1 (Satb1),        miR-632, or a combination thereof;    -   iii) at least a 20-fold increase in the expression of one or        more of the rat ES cell-specific genes as set forth in Table 14        when compared to a F1H4 mouse ES cell;    -   iv) at least a 20-fold increase in the expression of at least 2,        3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25 or more of the rat ES cell-specific genes as        set forth in Table 14 when compared to a F1H4 mouse ES cell;    -   v) the expression of one or more of rat ES cell-specific genes        as set forth in Table 13;    -   vi) the expression of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,        40, 45, 50 or more of the rat ES cell-specific genes as set        forth in Table 13;    -   vii) at least a 20-fold increase in the expression of one or        more of the rat ES cell-specific genes as set forth in Table 13        when compared to a F1H4 mouse ES cell;    -   viii) at least a 20-fold increase in the expression of at least        2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,        20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more of the rat ES        cell-specific genes as set forth in Table 13 when compared to a        F1H4 mouse ES cell;    -   ix) at least a 20-fold decrease in the expression of one or more        of the rat ES cell-specific genes as set forth in Table 12 when        compared to a F1H4 mouse ES cell; and/or    -   x) at least a 20-fold decrease in the expression of at least 2,        3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more of the rat ES        cell-specific genes as set forth in Table 12 when compared to a        F1H4 mouse ES cell;    -   xi) any combination of expression of the rat ES cell-specific        genes of parts (i)-(x);    -   xii) a relative expression level of pluripotency markers as        shown in Table 15 for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17 or 18 of the listed pluripotency markers.        See, pluripotency ranking column of Table 15 for relative        expression levels;    -   xiii) a relative expression level of the mesodermal markers as        shown in Table 15 for at least 2, 3, or 4 of the listed        mesodermal markers. See, mesodermal ranking column in Table 15        for relative expression levels;    -   xiv) a relative expression level of endodermal markers as shown        in Table 15 for at least 2, 3, 4, 5, or 6 of the listed        endodermal markers. See, endodermal ranking column in Table 15        for relative expression levels;    -   xv) a relative expression level of neural markers as shown in        Table 15 for at least 2 and 3 of the listed neural markers. See,        neural ranking column in table 15 for relative expression        levels;    -   xvi) a relative expression level of trophectoderm markers as        shown in Table 15 for the listed trophectoderm markers. See,        trophectoderm ranking column in table 15 for relative expression        levels;    -   xvii) any relative expression level of one or more (2, 3, 4, 5,        6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,        23, 24, 25, 26, 27, 28, 29 or 30) of the pluripotency markers,        mesodermal markers, endodermal markers, neural markers and/or        trophectoderm markers set forth in Table 15;    -   xviii) the relative expression level of each of the markers set        forth in table 15;    -   xix) any combination of the signatures set forth in xii-xiix;        and/or    -   xx) any combination of the signature set forth in i-xiix.

(i) have the ability to produce a F0 rat;

(j) capable of being subcultured and maintaining the undifferentiatedstate;

(k) having the same number of chromosomes a normal rat cell;

(l) maintain pluripotency in vitro without requiring paracrine LIFsignaling; and/or

(m) have self renewal, meaning they divide indefinitely whilemaintaining pluripotency.

iii. Expression Cassettes

The terms “polynucleotide,” “polynucleotide sequence,” “nucleic acidsequence,” and “nucleic acid fragment” are used interchangeably herein.These terms encompass nucleotide sequences and the like. Apolynucleotide may be a polymer of RNA or DNA that is single- ordouble-stranded, that optionally contains synthetic, non-natural oraltered nucleotide bases. A polynucleotide in the form of a polymer ofDNA may be comprised of one or more segments of cDNA, genomic DNA,synthetic DNA, or mixtures thereof. Polynucleotides can comprisedeoxyribonucleotides and ribonucleotides include both naturallyoccurring molecules and synthetic analogues, and any combination these.The polynucleotides provided herein also encompass all forms ofsequences including, but not limited to, single-stranded forms,double-stranded forms, hairpins, stem-and-loop structures, and the like.

Further provided are recombinant polynucleotides. The terms “recombinantpolynucleotide” and “recombinant DNA construct” are used interchangeablyherein. A recombinant construct comprises an artificial or heterologouscombination of nucleic acid sequences, e.g., regulatory and codingsequences that are not found together in nature. In other embodiments, arecombinant construct may comprise regulatory sequences and codingsequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. Such aconstruct may be used by itself or may be used in conjunction with avector. If a vector is used, then the choice of vector is dependent uponthe method that is used to transform the host cells as is well known tothose skilled in the art. For example, a plasmid vector can be used.Genetic elements required to successfully transform, select andpropagate host cells and comprising any of the isolated nucleic acidfragments provided herein. Screening may be accomplished by Southernanalysis of DNA, Northern analysis of mRNA expression, immunoblottinganalysis of protein expression, or phenotypic analysis, among others.

In specific embodiments, one or more of the components described hereincan be provided in an expression cassette for expression in a rat cell.The cassette can include 5′ and 3′ regulatory sequences operably linkedto a polynucleotide provided herein. “Operably linked” means afunctional linkage between two or more elements. For example, anoperable linkage between a polynucleotide of interest and a regulatorysequence (i.e., a promoter) is a functional link that allows forexpression of the polynucleotide of interest. Operably linked elementsmay be contiguous or non-contiguous. When used to refer to the joiningof two protein coding regions, operably linked means that the codingregions are in the same reading frame. In another instance, a nucleicacid sequence encoding a protein may be operably linked to regulatorysequences (e.g., promoter, enhancer, silencer sequence, etc.) so as toretain proper transcriptional regulation. The cassette may additionallycontain at least one additional polynucleotide of interest to beco-introduced into the rat ES cell. Alternatively, the additionalpolynucleotide of interest can be provided on multiple expressioncassettes. Such an expression cassette is provided with a plurality ofrestriction sites and/or recombination sites for insertion of arecombinant polynucleotide to be under the transcriptional regulation ofthe regulatory regions. The expression cassette may additionally containselection marker genes.

The expression cassette can include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a recombinant polynucleotide provided herein, and atranscriptional and translational termination region (i.e., terminationregion) functional in mammalian cell or a host cell of interest. Theregulatory regions (i.e., promoters, transcriptional regulatory regions,and translational termination regions) and/or a polynucleotide providedherein may be native/analogous to the host cell or to each other.Alternatively, the regulatory regions and/or a polynucleotide providedherein may be heterologous to the host cell or to each other. Forexample, a promoter operably linked to a heterologous polynucleotide isfrom a species different from the species from which the polynucleotidewas derived, or, if from the same/analogous species, one or both aresubstantially modified from their original form and/or genomic locus, orthe promoter is not the native promoter for the operably linkedpolynucleotide. Alternatively, the regulatory regions and/or arecombinant polynucleotide provided herein may be entirely synthetic.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked recombinantpolynucleotide, may be native with the host cell, or may be derived fromanother source (i.e., foreign or heterologous) to the promoter, therecombinant polynucleotide, the host cell, or any combination thereof.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation. Toward this end, adapters or linkers may be employed tojoin the DNA fragments or other manipulations may be involved to providefor convenient restriction sites, removal of superfluous DNA, removal ofrestriction sites, or the like. For this purpose, in vitro mutagenesis,primer repair, restriction, annealing, resubstitutions, e.g.,transitions and transversions, may be involved.

A number of promoters can be used in the expression cassettes providedherein. The promoters can be selected based on the desired outcome. Itis recognized that different applications can be enhanced by the use ofdifferent promoters in the expression cassettes to modulate the timing,location and/or level of expression of the polynucleotide of interest.Such expression constructs may also contain, if desired, a promoterregulatory region (e.g., one conferring inducible, constitutive,environmentally- or developmentally-regulated, or cell- ortissue-specific/selective expression), a transcription initiation startsite, a ribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

iv. Generating F0 Rat Embryos and F1 Progeny Having the Targeted GeneticModification

The various methods and compositions provided herein can be used togenerate a genetically modified rat. Such methods generally comprise (a)introducing into the genome of an isolated rat ES cell disclosed hereina targeted genetic modification to form a rat ES cell having a geneticmodification; (b) implanting at least one of the genetically modifiedrat ES cells having the genetic modification into a rat host embryo toproduce a F0 embryo; (c) implanting the F0 embryo into a surrogatemother; (d) gestating the F0 embryo in the surrogate mother to term;and, (e) identifying a F0 rat having the genetic modification.

The genetically modified rat ES cells having the genetic modificationcan be implanted into a rat host embryo that is from the same rat strainor from a different rat strain. For example, a genetically modified DArat ES cell can be implanted into a DA rat host embryo or it can beimplanted into an SD host embryo, ACI host embryo or other heterologousrat host embryo. Similarly, a genetically modified ACI rat ES cell canbe introduced into an ACI rat host embryo or it can be introduced intoan SD host embryo, DA host embryo or other heterologous rat host embryo.Likewise, the surrogate mother can be from the same rat strain as thegenetically modified rat cell and/or the rat host embryo or thesurrogate mother can be from a different rat strain as the geneticallymodified rat cell and/or the rat host embryo. In one non-limitingembodiment, the genetically modified rat cell is from a DA strain, thehost rat embryo is from an SD host embryo and the surrogate mother isfrom a DA strain. In another non-limiting embodiment, the geneticallymodified rat cell is from an ACI strain, the host rat embryo is from anSD strain, and the surrogate mother is from a DA strain.

In still further embodiments, the chimeric rat (F0) can be breed toproduce an F1 progeny that is heterozygous for the targeted geneticmodification. In addition, the male rat of the F1 progeny can be breedwith a female rat of the F1 progeny to obtain an F2 progeny that ishomozygous for the genetic modification.

The methods and compositions provided herein allow for at least 1%, 3%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%or greater of the F0 F0 rats having the genetic modification to transmitthe genetic modification to the F1 progeny. In some embodiments, the ratES cells having the targeted genetic modification are introduced into apre-morula stage embryo from a corresponding organism, e.g., an 8-cellstage mouse embryo. See, e.g., U.S. Pat. Nos. 7,576,259, 7,659,442,7,294,754, and US 2008-0078000 A1, all of which are incorporated byreference herein in their entireties. In other embodiments, the For, thedonor rat ES cells may be implanted at 4 cell stage, 8 cell stage of thehost embryo.

The rat embryos comprising the genetically modified rat ES cells isincubated until the blastocyst stage and then implanted into a surrogatemother to produce an F0. Rats bearing the genetically modified genomiclocus can be identified via modification of allele (MOA) assay asdescribed herein. The resulting F0 generation derived from thegenetically modified rat ES cells is crossed to a wild-type rat toobtain F1 generation offspring. Following genotyping with specificprimers and/or probes, F1 rats that are heterozygous for the geneticallymodified genomic locus are crossed to each other to produce a rat thatis homozygous for the genetically modified genomic locus.

Further provided is a F0 rat embryo comprising an inner cell mass havingat least one heterologous stem cell comprising any one of the rat EScells provided herein. In other embodiments, progeny of a rat F0 embryoare provided wherein at least 50%, 60%, 70% or more of the F0 progenyare derived from a genetically modified rat ES cell of as disclosedherein.

In one aspect, a method for making a rat ES cell is provided, comprisingderiving from a morula-stage rat embryo, a blastocyst-stage rat embryo,or a rat embryo at a developmental stage between a morula-stage embryoand a blastocyst-stage embryo a rat cell, and culturing the rat cellfrom the rat embryo under conditions sufficient to maintain pluripotencyand/or totipotency. In on embodiment, the conditions sufficient tomaintain pluripotency and/or totipotency include 2i media.

In one aspect, a method for making a genetically modified rat isprovided, comprising a step of modifying a rat ES cell genome with anucleic acid sequence of interest to form a modified rat ES cell, andemploying the modified rat ES cell as a donor rat ES cell, combining therat donor ES cell with a rat host embryo, culturing the donor ES celland rat host embryo, and employing the cultured host embryo to make agenetically modified rat.

In one aspect, a method for making genetically modified rat F1 progenyis provided, comprising a step of modifying a rat ES cell genome with anucleic acid sequence of interest to form a modified rat ES cell, andemploying the modified rat ES cell as a donor rat ES cell, combining therat donor ES cell with a rat host embryo, culturing the donor ES celland rat host embryo, and employing the cultured host embryo to make agenetically modified rat, wherein the progeny are about 3%, about 10% ormore, or about 63% or more derived from the genetically modified donorrat ES cell.

In one embodiment, the cultured host embryo is implanted into asurrogate rat mother, and the cultured host embryo is gestated in thesurrogate mother.

In one aspect, a method of transmitting a genetic modification from arat pluripotent cell to a rat progeny with high frequency is provided,comprising genetically modifying a pluripotent rat cell with a nucleicacid sequence of interest on a bacterial artificial chromosome to form agenetically modified rat pluripotent cell, and employing the geneticallymodified rat pluripotent cell with a rat host embryo in a rat surrogatemother to generate a progeny comprising the genetic modification and,optionally, breeding the progeny.

In one aspect, a method for making a rat ES cell is provided, whereinthe method comprises culturing a frozen 8-cell stage embryo to ablastocyst stage, and deriving from the cultured blastocyst a rat cell,and culturing the rat cell under conditions sufficient to maintainpluripotency and/or totipotency.

V. Variants, Fragments and Sequence Identity

Active variants and fragments of the disclosed LIF polypeptide,particularly the mouse LIF polypeptide are provided herein. “Variants”refer to substantially similar sequences. As used herein, a “variantpolypeptide” is intended to mean a polypeptide derived from the nativeprotein by deletion (so-called truncation) of one or more amino acids atthe N-terminal and/or C-terminal end of the native protein; deletionand/or addition of one or more amino acids at one or more internal sitesin the native protein; or substitution of one or more amino acids at oneor more sites in the native protein. Variant polypeptides continue topossess the desired biological activity of the native polypeptide, thatis, they inhibit the differentiation of rat and/or mouse embryonic stemcells and contribute to stem cell self-renewal. A variant of apolypeptide or disclosed herein (i.e. SEQ ID NOS: 1 or SwissProtAccession No. P09056) will typically have at least about 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity with the reference sequence.

The term “fragment” refers to a portion of an amino acid comprising aspecified number of contiguous amino acid. In particular embodiments, afragment of a polypeptide disclosed herein may retain the biologicalactivity of the full-length polypeptide and hence inhibit thedifferentiation of rat and/or mouse embryonic stem cells and contributeto stem cell self-renewal. Fragments of a polypeptide sequence disclosedherein (i.e. SEQ ID NOS: 1 or SwissProt Accession No. P09056) maycomprise at least 10, 15, 25, 30, 50, 60, 70, 80, 90, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, contiguous amino acids, or up tothe total number of amino acids present in a full-length protein.

As used herein, “sequence identity” or “identity” in the context of twopolynucleotides or polypeptide sequences makes reference to the residuesin the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. “Equivalentprogram” means any sequence comparison program that, for any twosequences in question, generates an alignment having identicalnucleotide or amino acid residue matches and an identical percentsequence identity when compared to the corresponding alignment generatedby GAP Version 10.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Rat ES Cell Derivation

rESC Characterization. As shown in FIG. 1, rESC grow as compactspherical colonies which routinely detach and float in the dish(close-up, FIG. 4). B, C, F, G: rESC express pluripotency markersincluding Oct-4 (FIG. 2A) and Sox2 (FIG. 2B), and express high levels ofalkaline phosphatase (FIG. 3, left panel). Karyotype for line DA.2B is42X,Y (FIG. 3, right panel). rESC often become tetraploid; thus, lineswere pre-screened by counting metaphase chromosome spreads; lines withmostly normal counts were then formally karyotyped. ACI blastocysts werecollected from super-ovulated females obtained commercially; DA blastswere cultured from frozen 8-cell embryos obtained commercially. Zonapellucidae were removed with Acid Tyrodes and blasts were plated ontomitotically inactivated MEFs. Outgrowths were picked and expanded usingstandard methods. All blasts were plated, cultured and expanded using 2imedia (Li et al. (2008) Germline competent embryonic stem cells derivedfrom rat blastocysts, Cell 135:1299-1310; incorporated herein byreference).

TABLE 3 Rat ES Cell Derivation ACI DA Blastocysts Frozen 8-cell embryosEmbryo source (Superovulation) cultured to blastocyst Blastocystsplated: 107 22 Outgrowths: 32 (30% of blasts) 10 (45% of blasts) Lines:16 (50% of 9 (90% of outgrowths) outgrowths) Karyotyped: 3; all 42X,Y 6:3 42X,X 3 42X,Y GLT validated: 1 (ACI.G1) 1 42X,X (DA.2C) 1 42X,Y(DA.2B)

Example 2 Rat Production

Chimeric rats were produced by blastocyst injection and transmission ofthe rESC genome through the germline. Chimeras produced by blastocystmicroinjection using parental ACI.G1 rESC are shown in FIG. 5. F1 agoutipups with albino littermates, sired by the ACI/SD chimera labeled withan asterisk (*) in FIG. 5 are shown in FIG. 6.

Germline transmission of parental rESC. Three euploid rESC lines wereevaluated for pluripotency by microinjection into albino SD blastocysts.Chimeras were identified by agouti coat color indicating rESCcontribution. For each line, a majority of chimeras transmitted the rESCgenome to F1 offspring (Table 4).

TABLE 4 Germline Transmission of Parental rESC Total pups rESC- GLTChimeras Germline from GLT derived efficiency Line bred transmitterschimeras pups (%) ACI.G1 5 3 (60%) 103 11 11 DA.2B 5 4 (80%) 129 11 9DA.2C 3 2 (66%) 45 7 16 (XX)

Example 3 rESC Targeting: The Rat Rosa 26 Locus

The rRosa26 locus lies between the Setd5 and Thumpd3 genes as in mouse,with the same spacing. The rRosa 26 locus (FIG. 7, Panel B) differs fromthe mRosa 26 locus (FIG. 7, Panel A). The mRosa26 transcripts consist of2 or 3 exons. The rat locus contains a 2nd exon 1 (Ex1b) in addition tothe homologous exon to mouse exon1 (Ex1a). No 3rd exon has beenidentified in rat. Targeting of a rRosa26 allele is depicted in FIG. 7,Panel C, where homology arms of 5 kb each were cloned by PCR usinggenomic DNA from DA rESC. The targeted allele contains a SA-lacZ-hUb-neocassette replacing a 117 bp deletion in the rRosa26 intron.

Targeting efficiency at the rRosa 26 locus was determined (Table 5).Linearized vector was electroporated into DA or ACI rESC, andtransfected colonies were cultured in 2i media+G418, using standardtechniques. Individual colonies were picked and screened using a Loss ofAllele (LOA) assay (Valenzuela, D. et al. (2003) High-throughputengineering of the mouse genome coupled with high-resolution expressionanalysis, Nature Biotech. 21:652-660, incorporated herein by reference).

TABLE 5 rRosa26 Targeting Efficiency Colonies Reconfirmed Targetingefficiency Cell line picked positives (%) DA.2B 192 4 2.1 ACI.G1 96 44.2

Chimera production and germline transmission using targeted Rosa26 rESC.Reconfirmed targeted rRosa26 clones were microinjected into SDblastocysts, which were then transferred to pseudopregnant DS recipientfemales, using standard techniques. Chimeras were identified by coatcolor; male F0 chimeras were bred to SD females. Germline (agouti) F1pups were genotyped for the presence of the targeted Rosa26 allele; nineof 22 agouti pups genotyped as heterozygous at the Rosa26 locus (Table6).

TABLE 6 Germline Transmission Using Targeted Rosa26 rESC ESC- R26 ClonesGermline rESC- derived clones producing Transmitting Total derived pupsCell line injected Chimeras Clones Pups Pups (%) DA.2B 4 3 2 AH7: 64AH7: 41 AH7: 63 AE3: 112 AE3: 6 AE3: 3 ACI.G1 4 4 1 DE9: 39 DE9: 4 10

Example 3 Derivation of Rat Embryonic Stem Cells

-   Superovulation Protocol, Rats-   Day 0: injected with pregnant mare serum: IP, 20 U (0.4 ml).-   Day 1: no action-   Day 2: (46 hr. later): injected with hCG, IP, 50 U (1 ml).

set up single female matings.

-   Day 3: checked plugs. Females were plugged. This is day 0.5.-   Day 6 (e3.5): Euthanized females and flushed embryos.    ES Cell Derivation Protocol (Superovulation)    Day 0:    -   1) Euthanized female rat with CO₂.    -   2) Swabbed ventral abdomen with 70% ethanol; using scissors,        opened the ventral body wall to expose the viscera.    -   3) Dissected out the oviducts and uterine horns and placed them        into a tissue culture dish containing warm N2B27 media. Washed        out as much blood as possible and transferred to a new dish with        N2B27.    -   4) Using a 1 ml syringe and a blunt 27 g needle, flushed media        through the uterine horns and oviducts to eject blastocysts into        the media.    -   5) Collected the blastocysts with a mouth pipet and transfer to        embryo culture dish containing KSOM+2i (1 μMPD0325901, 3 μM        CHIR99021). KSOM is a culture medium produced by Millipore.        Catalog number is MR-106-D.    -   6) Cultured overnight at 37°; 7.5% CO₂.        ES Cell Derivation Protocol (Frozen Embryos)        Day 0:    -   1) Thawed frozen 8-cell embryos (commercially obtained) into M2        medium. Cultured 10 minutes at room temperature.    -   2) Transferred to KSOM+2i and culture overnight.        ES Cell Derivation Protocol (Same for Both)        Day 1:    -   1) Transferred cavitated embryos to 2i medium & culture        overnight.    -   2) Continued culturing un-cavitated embryos in KSOM+2i        Day 2:    -   1) Transferred all remaining embryos to 2i medium (whether or        not they've cavitated).    -   2) Cultured overnight; continued culturing earlier embryos in 2i        medium.        Day 3:    -   1) Transferred embryos for 30-60 seconds with Acid Tyrodes to        remove the zona pellucida.    -   2) Washed embryos 3× in 2i medium to remove Acid Tyrodes.    -   3) Deposited each embryo into a separate well of a 96-well        feeder plate (the well contains a monolayer of mitotically        inactivated mouse embryonic fibroblasts (MEFs).    -   4) Cultured overnight in 2i medium.        Day 4-5:    -   1) Monitored plated embryos for the presence of an outgrowth (an        amorphous undifferentiated mass of cells). Outgrowths are ready        for transfer when they are approximately twice the size of the        plated embryo.    -   2) Each day: remove spent media with a mircropipet and replace        with fresh 2i media.    -   3) Transferred outgrowths to new feeder wells:        -   a. Removed spent media and gently wash well with PBS.        -   b. Removed PBS and add 30 μl 0.05% trypsin; incubate for 10            minutes.        -   c. Stopped trypsin reaction by adding 30 μl 2i+10% FBS.        -   d. Gently dissociated the cells with a micropipettor and            transferred entire contents of the well to a new well in a            24-well feeder plate. This was Passage 1 (P1).    -   e. Cultured overnight in 2i medium.        Day 5-8: (timing depends on how fast each line expands)    -   1) Changed media each day (2i media) and monitored for the        presence of colonies with an ESC morphology.    -   2) When colonies appear, continued culturing until colonies        expand to ˜50% confluency.    -   3) Tryspinzed and passaged colonies as before; plated on        feeders, 1 well per line, in a 6-well dish. This was Passage 2        (P2).        Ongoing:    -   1) Continued feeding and monitoring each line until        approximately 50% confluent.    -   2) Trypsinized cells as usual.    -   3) stopped trypsin with 2i+10% FBS; pelleted the cells by        centrifugation (5′, 1200 rpm in Beckman-Coulter tabletop        centrifuge).    -   4) Aspirated the supernatant and gently resuspend the cells in        400 μl Freezing Medium (70% 2i, 20% FBS, 10% DMSO).    -   5) Distributed the cells into 2 vials and freeze at −80°. This        was Passage 3 (P3).    -   6) For long-term storage, transferred the vials to liquid N2        storage.        The 2i media was prepared as follows in Table 7.

Reagent Vendor Concentration DMEM/F12 basal media Invitrogen/LifeTechnologies 1x Neurobasal media Invitrogen/Life Technologies 1xPenicillin/streptomycin Invitrogen/Life Technologies 1% L-GlutamineInvitrogen/Life Technologies  4 mM 2-Mercaptoethanol Invitrogen/LifeTechnologies  0.1 mM N2 supplement Invitrogen/Life Technologies 1x B27supplement Invitrogen/Life Technologies 1x LIF Millipore 100 U/mlPD0325901 (MEK Stemgent  1 uM inhibitor). CHIR99021 (GSK Stemgent  3 uMinhibitor).Materials: Pregnant Mare's Serum Gonadotropin (PMSG)

Human Pregnancy Urine Chorionic Gonadotropin (HCG)

Female Rats (5-12 weeks old)

Male rats (12 wks. to 8 mos. old), one per cage

Syringes/needles

Animal room with lights on 6:00-18:00

Procedure:

Day 1: 8:00-10:00 AM

-   -   Inject females with 20 IU PMSG (0.4 ml), IP    -   Discard unused PMSG. 6 p Day 3: 8:00-10:00 AM (48 hours after        PMSG injection)    -   Inject females with 50 IU HCG (1 ml), IP    -   Place one female per male in mating cage.    -   Discard unused HCG.

Day 4: 8:00-10:00 AM (24 hrs. after HCG injection)

-   -   Check females for plugs.        Hormone Suppliers

PMSG: Sigma #G-4877 (1000 IU). Resuspend in PBS to a final [ ] of 50IU/ml. Store at −20° in 1 ml aliquots.

HCG: Sigma #CG-5 (5000 IU). Resuspend in PBS to a final [ ] of 50 IU/ml.Store at −20° in 1 ml aliquots.

Example 4 Karyotyping of Rat Embryonic Stem Cell Lines

The rat ES cell line generated herein were karyotyped, and the resultsare summarized in

Tables 8-11.

TABLE 8 ACI.G1 Karyotyping Results Number of cells Number of cellskaryotyped 7 Number of cells analyzed 20 Number of 42, XY cells 18Number of abnormal cells 2 40, XY, −5, −9 1 41, XY, −14 1 42, XY 18Other notes: Two analyzed cells were missing different autosomes, whichmay be a sporadic occurrence due to technical artifact. 90% of analyzedcells had a normal male 42, XY karyotype. FIG. 9 provides a photographshowing the analysis of the chromosome number of the ACI.G1 rat ES cellline.

TABLE 9 DA.2B Karyotyping Results Number of cells Number of cellskaryotyped 6 Number of cells analyzed 20 Number of 42, XY cells 20Number of abnormal cells 0 42, XY 20 Other notes: All analyzed cells hada normal diploid 42, XY karyotype. FIG. 10 provides a photograph showingthe analysis of the chromosome number of the DA.2B rat ES cell line.

TABLE 10 DA.C2 Karyotyping Results Number of cells Number of cellskaryotyped 5 Number of cells analyzed 20 Number of 42, XY cells 20Number of abnormal cells 0 42, XX Other notes: 100% of analyzed cellshad normal female XX rat karyotype. FIG. 11 provides a photographshowing the analysis of the chromosome number of the DA.C2 rat ES cellline.

TABLE 11 Blastocysts Lines Lines Strain plated established KaryotypedKaryotypes BN × SD 41 8 (20%) 5 all lines were high % complex polyploidF1 ACI 27 16 (60%)  3 G1: 90% 42 XY; others were 70-85% euploid DA 20 9(45%) 6 2B: 100% 42 XY; 2C: 100% 42 XX; others were 95-100% euploid F3444 1 (25%) 0 Totals 92 34 (37%) 

Example 5 Electroporation of Vector into Rat Embryonic Stem Cell

1. Passaged rat ES cells 24-48 hrs prior to electroporation.

2. Changed media to RVG2i+ROCKi (10 μM Y-27632) 24 hr. prior toelectroporation

3. Changed media 30′ prior to trypsinization.

4. Aliquoted DNA to be electroporated.

5. Allowed DNA to warm at RT for >10 min.

6. Heated DNA for 5′ @ 62° C. Place DNA on ice.

7. Trypsinized cells:

-   -   a. Collected floating colonies. Washed plate to collect as many        floaters as possible.    -   b. Pelleted colonies: 3′ @ 750 rpm.    -   c. Washed pellet 1× with 5-10 ml PBS and re-spin/pellet    -   d. Aspirated supernatant; add 500λ, trypsin, 0.05%+1% chicken        serum.        -   i. Did not pool more than 1 10 cm plate of colonies per            tube. If there are too many colonies packed into the bottom            of the tube during trypsinization they will clump and most            of the cells will be lost.    -   e. 4′ @ 37°. Pipeted colonies several times to minimize        clumping.    -   f. Repeated steps 1-2×: 4′ @ 37°.    -   g. Stopped trypsin with 500λ, RVG2i+10% FBS.

8. Pelleted cells: 5′ @ 1200 rpm.

9. Resuspend cells in 10 ml PBS. Count two 20λ, aliquots to determinetotal cell number.

10. Pelleted cells (5′/1200 rpm); calculate total cell number and totalresuspension volume to achieve correct cell concentration (target #/75μl EP buffer).

11. Resuspend in a minimal volume of EP buffer; measure total volume andadjust to target volume with EP buffer. Electroporation buffer is soldby Millipore. The catalog # is ES-003-D. See, Valenzuela et al. (2003)Nature Biotechnology 21:652-659, which is herein incorporated byreference.

12. Add 75λ, cells to 50λ, DNA; transfer the 125λ, cells/DNA solution toone well of a BTX 48-well cuvette.

-   -   a. Filled the empty wells in the same column with 125λ, EP        buffer.

13. Pulsed the cuvette once in the BTX electroporator:

-   -   a. Settings: 400V; Ω; 100 μF (settings may vary)

14. Placed cuvette on ice for 15′ to recover.

15. Removed cells into 5 ml RVG2i+1004 ROCKi.

16. Added to 15 cm plate with 20 ml RVG2i+1004 ROCKi. Plate has 2× neoRMEFs (or other MEFs depending on project). The neoR selectable marker isthe neomycin phosphotransferase (neo) gene of Beck et al. (1982) Gene,19:327-36 or in U.S. Pat. Nos. 7,205,148 or 6,596,541, each of which areherein incorporated by reference.

17. Incubated @ 37°. Begin selection 48 hrs later.

ROCK inhibitor used was Y-27632.

Example 6 Selecting Targeted Genetic Modification in a Rat EmbryonicStem Cell

1. Passaged cells for 24-48 hrs prior to electroporation.

2. Changed media to RVG2i+ROCKi (10 μM Y-27632) 24 hr. prior toelectroporation

3. Changed media 30′ prior to trypsinization.

4. Aliquoted DNA to be electroporated.

5. Allowed DNA warm at RT for >10 min.

6. Heated DNA for 5′ @ 62° C. Place DNA on ice.

7. Trypsinized cells:

-   -   h. Collected floating colonies. Washed plate to collect as many        floaters as possible.    -   i. Pelleted colonies: 3′ @ 750 rpm.    -   j. Washed pellet 1× with 5-10 ml PBS and re-spin/pellet    -   k. Aspirated supernatant; add 500λ, trypsin, 0.05%+1% chicken        serum.        -   i. Did not pool more than 1 10 cm plate of colonies per            tube. If there are too many colonies packed into the bottom            of the tube during trypsinization they will clump and most            of the cells will be lost.    -   l. 4′ @ 37°. Pipeted colonies several times to minimize clumping    -   m. Repeated 1-2×: 4′ @ 37°.    -   n. Stopped trypsin with 500λ, RVG2i+10% FBS.

8. Pelleted cells: 5′ @ 1200 rpm.

9. Resuspended cells in 10 ml PBS. Count two 20λ, aliquots to determinetotal cell number.

10. Pelleted cells (5′/1200 rpm); calculate total cell number and totalresuspension volume to achieve correct cell concentration (target #/75μl EP buffer).

11. Resuspend in a minimal volume of EP buffer; measured total volumeand adjusted to target volume with EP buffer. 6 p 12. Added 75λ, cellsto 50λ DNA; transfer the 125λ cells/DNA solution to one well of a BTX48-well cuvette.

-   -   a. Filled the empty wells in the same column with 125λ EP        buffer.

13. Pulsed the cuvette once in the BTX electroporator:

-   -   a. Settings: 400V; 400V; Ω; 100 μF (settings may vary)

14. Placed cuvette on ice for 15′ to recover.

15. Removed cells into 5 ml RVG2i+10 μM ROCKi.

16. Added to 15 cm plate with 20 ml RVG2i+1004 ROCKi. Plate had 2× neoRMEFs (or other MEFs depending on project).

17. Incubated @ 37°. Began selection 48 hrs later.

18. G418 selection protocol was as follows:

-   -   a. Day 2 (2^(nd) day after EP): incubated cells in 2i        media+G418, 75 μg/ml.    -   b. Day 3: incubated cells in 2i media without G418    -   c. Day 4: incubated cells in 2i media+G418, 75 μg/ml.    -   d. Day 5: incubated cells in 2i media without G418    -   e. Day 6: incubated cells in 2i media+G418, 75 μg/ml.    -   f. Day 7: incubated cells in 2i media without G418    -   g. Day 8: incubated cells in 2i media+G418, 75 μg/ml.    -   h. Day 9: incubated cells in 2i media without G418    -   i. Day 10: incubated cells in 2i media+G418, 75 μg/ml.    -   j. Day 11: incubated cells in 2i media without G418    -   k. Day 12: picked colonies to expand for screening. Each colony        was dissociated in 0.05% trypsin+1% chicken serum for 10 minutes        and then plated into 1 well of a 96-well feeder plate.

19. Expanded colonies for 3 days in 2i media.

20. Passaged clones 1:1 to new 96-well feeder plates.

21. Expanded clones for 3 days in 2i media.

22. For each clone, dissociated colonies in trypsin. Froze 2/3 of eachclone and store at −80°; plated the remaining 1/3 onto laminin plates(96-well plates coated with 10 μg/ml laminin).

23. When the laminin plates were confluent, passed off to the screeninglab for genotyping of the clones.

Example 7 Molecular Signature of the Rat Embryonic Stem Cells

The genes listed in Table 13 were expressed at levels 20-fold higher inrat ES cells than the corresponding genes in mouse ES cells. The geneslisted in Table 12 were expressed at 20-fold lower in rat ES cells thanthe corresponding genes in mouse ES cells.

The microarray data in Tables 12 and 13 was generated as follows. Rat EScells (ACI.G2 and DA.2B) and mouse ES cells (F1H4) were cultured in 2imedia for 3 passages until confluent. F1H4 cells were cultured ongelatin-coated plates in the absence of feeders. F1H4 mouse ES cellswere derived from 129S6/SvEvTac and C57BL/6NTac heterozygous embryos(see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou, W. T., Auerbach, W.,Frendewey, D., Hickey, J. F., Escaravage, J. M., Esau, L., Dore, A. T.,Stevens, S., Adams, N. C., Dominguez, M. G., Gale, N. W., Yancopoulos,G. D., DeChiara, T. M., Valenzuela, D. M. (2007), incorporated byreference herein in its entirety).

The following protocol was used for sample prep:

Materials included TRIzol Reagentp; RNA Lysis Buffer (Zymo Kit); and 1.5mL Eppendorf tubes.

The 1.5 mL Eppendorf tubes were labeled with the Sample ID. Cells grownon a plate were rinsed in 37 C PBS. PBS was removed and 300 ul of Trizolwas added. A scraper was used to break the cells in Trizol. The lysedcells were collected in Trizol in a 1.5 mL Epperdorf tube. For cellsgrown on suspension, the cells were rinsed in 37 C PBS. The cells werecollected in a 1.5 mL tube, the cells were spun down, PBS was removedand 300 ul of Trizol was added. The cells were pipeted up and down tobreak the cells. Samples were sorted for FACS with 10¹ to 10⁵ cells, thevolume was concentrated to less than 100 uL. 4 volumes of RNA Lysisbuffer was added and mix by pipetting. For sample, 320 uL RNA Lysisbuffer was added to 80 uL sample. Samples were stored at −20° C.

RNA-Seq was used to measure the expression level of mouse and rat genes.Sequencing reads were mapped to mouse and rat reference genome byTophat, and RPKM (fragments per kilobase of exon per million fragmentsmapped) were calculated for mouse and rat genes. Homology genes based ongene symbol were selected, and then used t-test to compare gene'sexpression level between mouse and rat.

miR-632 is in the top 10 highest expressed in rat ESCs but they were notexpressed in mouse ES cells. There is therefore no comparative data forthese genes. Based on the levels of expression compared to other genesand their known function in the embryonic development, the expression ofmiR-632 were used as a marker for rat ES cells.

TABLE 12 The genes listed were expressed at levels 20-fold lower in ratES cells than the corresponding genes in mouse ES cells. ID Notes SymbolEntrez Gene Name Location Type(s) Drug(s) Abcb1b Abcb1b ATP-bindingPlasma transporter cassette, sub- Membrane family B (MDR/TAP), member 1BActa2 ACTA2 actin, alpha 2, Cytoplasm other smooth muscle, aorta Actg2ACTG2 actin, gamma 2, Cytoplasm other smooth muscle, enteric Aebp1 AEBP1AE binding protein Nucleus peptidase 1 Angptl2 ANGPTL2 angiopoietin-like2 Extracellular other Space Ankrd1 ANKRD1 ankyrin repeat Cytoplasmtranscription domain 1 (cardiac regulator muscle) Anxa1 ANXA1 annexin A1Plasma other hydrocortisone Membrane Anxa6 ANXA6 annexin A6 Plasma otherMembrane Anxa8 ANXA8L2 annexin A8-like 2 Plasma other (includes Membraneothers) Arhgef25 ARHGEF25 Rho guanine Cytoplasm other nucleotideexchange factor (GEF) 25 Axl AXL AXL receptor Plasma kinase cabozantinibtyrosine kinase Membrane Basp1 BASP1 brain abundant, Nucleustranscription membrane attached regulator signal protein 1 Bgn BGNbiglycan Extracellular other Space Bst2 BST2 bone marrow Plasma otherstromal cell antigen Membrane 2 Btf3 BTF3 basic transcription Nucleustranscription factor 3 regulator Btg2 BTG2 BTG family, Nucleustranscription member 2 regulator Capsl CAPSL calcyphosine-like Otherother Cav1 CAV1 caveolin 1, Plasma transmembrane caveolae protein,Membrane receptor 22 kDa Ccdc80 CCDC80 coiled-coil domain Nucleus othercontaining 80 Ccnd2 CCND2 cyclin D2 Nucleus other Cd248 CD248 CD248molecule, Plasma other endosialin Membrane Cd44 CD44 CD44 moleculePlasma enzyme (Indian blood Membrane group) Cd97 CD97 CD97 moleculePlasma G-protein Membrane coupled receptor Cdc42ep5 CDC42EP5 CDC42effector Cytoplasm other protein (Rho GTPase binding) 5 Cdh11 CDH11cadherin 11, type 2, Plasma other OB-cadherin Membrane (osteoblast)Cdkn2a CDKN2A cyclin-dependent Nucleus transcription kinase inhibitor 2Aregulator Cdo1 CDO1 cysteine Cytoplasm enzyme dioxygenase type 1 Clip3CLIP3 CAP-GLY domain Cytoplasm other containing linker protein 3 Cln5CLN5 ceroid- Cytoplasm other lipofuscinosis, neuronal 5 Cnn1 CNN1calponin 1, basic, Cytoplasm other smooth muscle Col1a1 COL1A1 collagen,type I, Extracellular other collagenase alpha 1 Space clostridiumhistolyticum Col1a2 COL1A2 collagen, type I, Extracellular othercollagenase alpha 2 Space clostridium histolyticum Col3a1 COL3A1collagen, type III, Extracellular other collagenase alpha 1 Spaceclostridium histolyticum Col5a2 COL5A2 collagen, type V, Extracellularother collagenase alpha 2 Space clostridium histolyticum Col6a2 COL6A2collagen, type VI, Extracellular other collagenase alpha 2 Spaceclostridium histolyticum Cryab CRYAB crystallin, alpha B Nucleus otherCsf1 CSF1 colony stimulating Extracellular cytokine factor 1 Space(macrophage) Cth CTH cystathionase Cytoplasm enzyme (cystathioninegamma-lyase) Cthrc1 CTHRC1 collagen triple Extracellular other helixrepeat Space containing 1 Ctsc CTSC cathepsin C Cytoplasm peptidaseCyr61 CYR61 cysteine-rich, Extracellular other angiogenic inducer, Space61 Ddx58 DDX58 DEAD (Asp-Glu- Cytoplasm enzyme Ala-Asp) box polypeptide58 Dkk3 DKK3 dickkopf WNT Extracellular cytokine signaling pathway Spaceinhibitor 3 Dmc1 DMC1 DNA meiotic Nucleus enzyme recombinase 1 Dpysl3DPYSL3 dihydropyrimidinase- Cytoplasm enzyme like 3 Dse DSE dermatansulfate Cytoplasm enzyme epimerase Dusp1 DUSP1 dual specificity Nucleusphosphatase phosphatase 1 Dusp27 DUSP27 dual specificity Otherphosphatase phosphatase 27 (putative) Dusp9 DUSP9 dual specificityNucleus phosphatase phosphatase 9 Ece2 ECE2 endothelin Plasma peptidaseconverting enzyme Membrane 2 Ecm1 ECM1 extracellular matrixExtracellular transporter protein 1 Space Egr1 EGR1 early growth Nucleustranscription response 1 regulator Emp1 EMP1 epithelial Plasma othermembrane protein Membrane 1 Emp3 EMP3 epithelial Plasma other membraneprotein Membrane 3 Ephx2 EPHX2 epoxide hydrolase Cytoplasm enzyme 2,cytoplasmic F3 F3 coagulation factor Plasma transmembrane activated III(thromboplastin, Membrane receptor recombinant tissue factor) humanfactor VII Fau FAU Finkel-Biskis- Cytoplasm other Reilly murine sarcomavirus (FBR-MuSV) ubiquitously expressed Fbn1 FBN1 fibrillin 1Extracellular other Space Fbxo15 FBXO15 F-box protein 15 Othertranscription regulator Fhl2 FHL2 four and a half Nucleus transcriptionLIM domains 2 regulator Flnc FLNC filamin C, gamma Cytoplasm other FosFOS FBJ murine Nucleus transcription osteosarcoma viral regulatoroncogene homolog Fundc2 FUNDC2 FUN14 domain Cytoplasm other containing 2Gjb3 GJB3 gap junction Plasma transporter protein, beta 3, Membrane 31kDa Gpa33 GPA33 glycoprotein A33 Plasma other (transmembrane) MembraneGpbp1l1 GPBP1L1 GC-rich promoter Other other binding protein 1- like 1Gpc3 GPC3 glypican 3 Plasma other Membrane Grb10 GRB10 growth factorCytoplasm other receptor-bound protein 10 Gstm1 GSTM5 glutathione S-Cytoplasm enzyme transferase mu 5 Hap1 HAP1 huntingtin- Cytoplasm otherassociated protein 1 Hist1h2bc HIST2H2BE histone cluster 2, Nucleusother (includes H2be others) Hmga2 HMGA2 high mobility Nucleus enzymegroup AT-hook 2 Hmgn3 Hmgn3 high mobility Nucleus other groupnucleosomal binding domain 3 Hormad1 HORMAD1 HORMA domain Nucleus othercontaining 1 Hsd17b14 HSD17B14 hydroxysteroid Cytoplasm enzyme (17-beta)dehydrogenase 14 Hspb1 HSPB1 heat shock 27 kDa Cytoplasm other protein 1Hspb8 HSPB8 heat shock 22 kDa Cytoplasm kinase protein 8 Htra1 HTRA1HtrA serine Extracellular peptidase peptidase 1 Space Ifi204 Ifi204interferon activated Nucleus transcription (includes gene 204 regulatorothers) Ifi44 IFI44 interferon-induced Cytoplasm other protein 44 Ifit1IFIT1B interferon-induced Cytoplasm other protein with tetratricopeptiderepeats 1B Ifitm3 IFITM2 interferon induced Cytoplasm othertransmembrane protein 2 Igf2 IGF2 insulin-like growth Extracellulargrowth factor 2 Space factor (somatomedin A) Igfbp7 IGFBP7 insulin-likegrowth Extracellular transporter factor binding Space protein 7 Il1rl1IL1RL1 interleukin 1 Plasma transmembrane receptor-like 1 Membranereceptor Inhba INHBA inhibin, beta A Extracellular growth Space factorInhbb INHBB inhibin, beta B Extracellular growth Space factor Irf7 IRF7interferon Nucleus transcription regulatory factor 7 regulator Isg15ISG15 ISG15 ubiquitin- Extracellular other like modifier Space Itga5ITGA5 integrin, alpha 5 Plasma transmembrane (fibronectin Membranereceptor receptor, alpha polypeptide) Jun JUN jun proto-oncogene Nucleustranscription regulator Junb JUNB jun B proto- Nucleus transcriptiononcogene regulator Lgals3bp LGALS3BP lectin, galactoside- Plasmatransmembrane binding, soluble, 3 Membrane receptor binding proteinLgals9 LGALS9 lectin, galactoside- Extracellular other binding, soluble,9 Space Lmna LMNA lamin A/C Nucleus other Lox LOX lysyl oxidaseExtracellular enzyme Space Loxl2 LOXL2 lysyl oxidase-like 2Extracellular enzyme Space Loxl3 LOXL3 lysyl oxidase-like 3Extracellular enzyme Space Lrp1 LRP1 low density Plasma transmembranelipoprotein Membrane receptor receptor-related protein 1 Mageb16 MAGEB16melanoma antigen Other other family B, 16 Mcam MCAM melanoma cell Plasmaother adhesion molecule Membrane Mgp MGP matrix Gla proteinExtracellular other Space Mmp2 MMP2 matrix Extracellular peptidasemarimastat metallopeptidase 2 Space (gelatinase A, 72 kDa gelatinase, 72kDa type IV collagenase) Mxra8 MXRA8 matrix-remodelling Other otherassociated 8 Myl9 MYL9 myosin, light chain Cytoplasm other 9, regulatoryMylpf MYLPF myosin light chain, Cytoplasm other phosphorylatable, fastskeletal muscle Nab2 NAB2 NGFI-A binding Nucleus transcription protein 2(EGR1 regulator binding protein 2) Ndufb4 NDUFB4 NADH Cytoplasmtransporter dehydrogenase (ubiquinone) 1 beta subcomplex, 4, 15 kDa Npm1NPM1 nucleophosmin Nucleus transcription (nucleolar regulatorphosphoprotein B23, numatrin) Nr0b1 NR0B1 nuclear receptor Nucleusligand- subfamily 0, group dependent B, member 1 nuclear receptor Nr4a1NR4A1 nuclear receptor Nucleus ligand- subfamily 4, group dependent A,member 1 nuclear receptor Nrp2 NRP2 neuropilin 2 Plasma kinase MembraneOas1a OAS1 2′-5′-oligoadenylate Cytoplasm enzyme synthetase 1, 40/46 kDaOasl2 Oasl2 2′-5′ oligoadenylate Other enzyme synthetase-like 2 P4ha2P4HA2 prolyl 4- Cytoplasm enzyme hydroxylase, alpha polypeptide II Parp3PARP3 poly (ADP-ribose) Nucleus enzyme polymerase family, member 3Pcolce PCOLCE procollagen C- Extracellular other endopeptidase Spaceenhancer Pcyt1b PCYT1B phosphate Cytoplasm enzyme cytidylyltransferase1, choline, beta Pdgfc PDGFC platelet derived Extracellular growthgrowth factor C Space factor Phlda1 PHLDA1 pleckstrin Cytoplasm otherhomology-like domain, family A, member 1 Phlda2 PHLDA2 pleckstrinCytoplasm other homology-like domain, family A, member 2 Pla2g1b PLA2G1Bphospholipase A2, Extracellular enzyme niflumic acid group IB Space(pancreas) Pla2g4a PLA2G4A phospholipase A2, Cytoplasm enzyme quinacrinegroup IVA (cytosolic, calcium- dependent) Porcn PORCN porcupine homologCytoplasm other (Drosophila) Postn POSTN periostin, Extracellular otherosteoblast specific Space factor Prrx1 PRRX1 paired related Nucleustranscription homeobox 1 regulator Prss23 PRSS23 protease, serine, 23Extracellular peptidase Space Psmb8 PSMB8 proteasome Cytoplasm peptidase(prosome, macropain) subunit, beta type, 8 Ptgs2 PTGS2 prostaglandin-Cytoplasm enzyme acetaminophen/ endoperoxide pentazocine, synthase 2acetaminophen/ (prostaglandin G/H clemastine/ synthase andpseudoephedrine, cyclooxygenase) aspirin/butalbital/ caffeine,acetaminophen/ caffeine/ dihydrocodeine, aspirin/ hydrocodone,aspirin/oxycodone, acetaminophen/ aspirin/caffeine, aspirin/pravastatin, acetaminophen/ dexbrompheniramine/ pseudoephedrine,aspirin/meprobamate, aspirin/caffeine/ propoxyphene, aspirin/butalbital/caffeine/codeine, aspirin/caffeine/ dihydrocodeine, chlorpheniramine/ibuprofen/ pseudoephedrine, licofelone, menatetrenone, icosapent,suprofen, lornoxicam, tiaprofenic acid, lumiracoxib, tenoxicam,naproxen/ sumatriptan, ibuprofen/ phenylephrine, acetaminophen/aspirin/codeine, naproxen/ esomeprazole, famotidine/ ibuprofen,ibuprofen/ phenylephrine/ chlorpheniramine, diclofenac/ misoprostol,acetaminophen/ butalbital/caffeine, hydrocodone/ ibuprofen,acetaminophen/ hydrocodone, acetaminophen/ tramadol, acetaminophen/codeine, acetaminophen/ oxycodone, acetaminophen/ propoxyphene, niflumicacid, nitroaspirin, ketoprofen, diclofenac, etoricoxib, naproxen,meclofenamic acid, pomalidomide, meloxicam, celecoxib, dipyrone,nimesulide, acetaminophen, mefenamic acid, diflunisal, ibuprofen,GW406381X, phenylbutazone, indomethacin, sulfasalazine, piroxicam,valdecoxib, aspirin, carprofen, zomepirac, rofecoxib, aspirin/methocarbamol, aspirin/caffeine/ orphenadrine, aspirin/ carisoprodol,aspirin/ carisoprodol/ codeine, acetaminophen/ butalbital, balsalazide,aspirin/ dipyridamole, acetaminophen/ butalbital/caffeine/ codeine,racemic flurbiprofen, phenacetin, sulindac, nabumetone, etodolac,tolmetin, ketorolac, oxaprozin, mesalamine, salsalate, fenoprofen,salicylic acid, acetaminophen/ chlorpheniramine/ hydrocodone/phenylephrine/ caffeine, bromfenac Ptn PTN pleiotrophin Extracellulargrowth Space factor Ptrf PTRF polymerase I and Nucleus transcriptiontranscript release regulator factor Rarg RARG retinoic acid Nucleusligand- etretinate, receptor, gamma dependent adapalene, nuclearisotretinoin, receptor tazarotene, acitretin, tretinoin, alitretinoin,fenretinide Rgs16 RGS16 regulator of G- Cytoplasm other proteinsignaling 16 Rn45s Rn45s 45S pre-ribosomal Other other RNA Rpl10a RPL10Aribosomal protein Other other L10a Rpl31 RPL31 ribosomal protein Otherother L31 Rpl37a RPL37A ribosomal protein Cytoplasm other L37a Rps10RPS10- RPS10-NUDT3 Cytoplasm other NUDT3 readthrough Rps14 RPS14ribosomal protein Cytoplasm translation S14 regulator Rps20 Rps20ribosomal protein Cytoplasm other S20 Rps26 RPS26 ribosomal proteinCytoplasm other S26 Rps9 RPS9 ribosomal protein Cytoplasm translation S9regulator S100a4 S100A4 S100 calcium Cytoplasm other binding protein A4S100a6 S100A6 S100 calcium Cytoplasm transporter binding protein A6Schip1 SCHIP1 schwannomin Cytoplasm other interacting protein 1 Sdc2SDC2 syndecan 2 Plasma other Membrane Serpine1 SERPINE1 serpin peptidaseExtracellular other drotrecogin alfa inhibitor, clade E Space (nexin,plasminogen activator inhibitor type 1), member 1 Serpine2 SERPINE2serpin peptidase Extracellular other inhibitor, clade E Space (nexin,plasminogen activator inhibitor type 1), member 2 Serpinf1 SERPINF1serpin peptidase Extracellular other inhibitor, clade F Space (alpha-2antiplasmin, pigment epithelium derived factor), member 1 Sh3gl2 SH3GL2SH3-domain Plasma enzyme GRB2-like 2 Membrane Slc19a2 SLC19A2 solutecarrier Plasma transporter family 19 Membrane (thiamine transporter),member 2 Slc25a5 SLC25A5 solute carrier Cytoplasm transporter clodronicacid family 25 (mitochondrial carrier; adenine nucleotide translocator),member 5 Slc29a1 SLC29A1 solute carrier Plasma transporter family 29Membrane (equilibrative nucleoside transporter), member 1 Slc35f2SLC35F2 solute carrier Other other family 35, member F2 Snrpn SNRPNsmall nuclear Nucleus other ribonucleoprotein polypeptide N Snx22 SNX22sorting nexin 22 Other transporter Sparc SPARC secreted protein,Extracellular other acidic, cysteine- Space rich (osteonectin) Spp1 SPP1secreted Extracellular cytokine phosphoprotein 1 Space Sult4a1 SULT4A1sulfotransferase Cytoplasm enzyme family 4A, member 1 Tagln TAGLNtransgelin Cytoplasm other Tcea3 TCEA3 transcription Nucleustranscription elongation factor A regulator (SII), 3 Tgfb3 TGFB3transforming Extracellular growth growth factor, beta Space factor 3Thbs1 THBS1 thrombospondin 1 Extracellular other Space Thbs2 THBS2thrombospondin 2 Extracellular other Space Tm4sf1 TM4SF1 transmembrane 4L Plasma other six family member Membrane 1 Tmbim1 TMBIM1 transmembraneCytoplasm other BAX inhibitor motif containing 1 Tmem176b TMEM176Btransmembrane Other other protein 176B Tnc TNC tenascin C Extracellularother Space Tpd52l1 TPD52L1 tumor protein D52- Cytoplasm other like 1Tpm2 TPM2 tropomyosin 2 Cytoplasm other (beta) Usp18 USP18 ubiquitinspecific Cytoplasm peptidase peptidase 18 Vim VIM vimentin Cytoplasmother Wfdc2 WFDC2 WAP four- Extracellular other disulfide core Spacedomain 2 Wisp2 WISP2 WNT1 inducible Extracellular growth signalingpathway Space factor protein 2 Ybx1 YBX1 Y box binding Nucleustranscription protein 1 regulator

TABLE 13 The genes listed were expressed at levels 20-fold higher in ratES cells than the corresponding genes in mouse ES cells. ID Notes SymbolEntrez Gene Name Location Type(s) Drug(s) Ajap1 Ajap1 adherens junctionOther other associated protein 1 Amd1 AMD1 adenosylmethionine Cytoplasmenzyme decarboxylase 1 Ankrd2 ANKRD2 ankyrin repeat Nucleustranscription domain 2 (stretch regulator responsive muscle) Arhgef9ARHGEF9 Cdc42 guanine Cytoplasm other nucleotide exchange factor (GEF) 9Atp5h Atp5h ATP synthase, H+ Cytoplasm enzyme transporting,mitochondrial F0 complex, subunit d Btg3 BTG3 BTG family, Nucleus othermember 3 Car6 CA6 carbonic anhydrase Extracellular enzyme methazolamide,VI Space hydrochlorothiazide, acetazolamide, trichloromethiazide,chlorothiazide, chlorthalidone, benzthiazide, sulfacetamide, topiramateCamk4 CAMK4 calcium/calmodulin- Nucleus kinase dependent protein kinaseIV Capn12 CAPN12 calpain 12 Other peptidase Cct6b CCT6B chaperoninCytoplasm transporter containing TCP1, subunit 6B (zeta 2) Cdx2 CDX2caudal type Nucleus transcription homeobox 2 regulator Cldn5 CLDN5claudin 5 Plasma other Membrane Clec3a CLEC3A C-type lectin Other otherdomain family 3, member A Clic6 CLIC6 chloride intracellular Plasma ionchannel channel 6 Membrane Dhrsx DHRSX dehydrogenase/ Other enzymereductase (SDR family) X-linked Dpysl2 DPYSL2 dihydropyrimidinase-Cytoplasm enzyme like 2 Dusp26 DUSP26 dual specificity Cytoplasm enzymephosphatase 26 (putative) Eci3 Eci3 enoyl-Coenzyme A Other enzyme deltaisomerase 3 Eef2k EEF2K eukaryotic Cytoplasm kinase elongation factor-2kinase Efna1 EFNA1 ephrin-A1 Plasma other Membrane Epha4 EPHA4 EPHreceptor A4 Plasma kinase Membrane Fank1 FANK1 fibronectin type IIINucleus transcription and ankyrin repeat regulator domains 1 Fhit FHITfragile histidine Cytoplasm enzyme triad Filip1 FILIP1 filamin ACytoplasm other interacting protein 1 Fmod FMOD fibromodulinExtracellular other Space Foxe1 FOXE1 forkhead box E1 Nucleustranscription (thyroid regulator transcription factor 2) Fry FRY furryhomolog Extracellular other (Drosophila) Space Gjb5 GJB5 gap junctionprotein, Plasma transporter beta 5, 31.1 kDa Membrane Gpx2 GPX2glutathione Cytoplasm enzyme peroxidase 2 (gastrointestinal) Grxcr2GRXCR2 glutaredoxin, Other other cysteine rich 2 Hecw2 HECW2 HECT, C2and WW Extracellular enzyme domain containing Space E3 ubiquitin proteinligase 2 Hey2 HEY2 hairy/enhancer-of- Nucleus transcription splitrelated with regulator YRPW motif 2 Icos Icos inducible T-cell co-Plasma other stimulator Membrane Ifitm1 IFITM1 interferon induced Plasmatransmembrane transmembrane Membrane receptor protein 1 Il1f8Interleukin 36 beta Il28ra Interleukin 28 receptor, alpha Igfbpl1IGFBPL1 insulin-like growth Other other factor binding protein-like 1Ipcef1 IPCEF1 interaction protein Cytoplasm enzyme for cytohesinexchange factors 1 Lctl Lctl lactase-like Cytoplasm other Ldhd LDHDlactate Cytoplasm enzyme dehydrogenase D Lef1 LEF1 lymphoid enhancer-Nucleus transcription binding factor 1 regulator Lefty1 LEFTY1left-right Extracellular growth determination factor Space factor 1 LifrLIFR leukemia inhibitory Plasma transmembrane factor receptor alphaMembrane receptor Lpar2 LPAR2 lysophosphatidic Plasma G-protein acidreceptor 2 Membrane coupled receptor Mog MOG myelin Extracellular otheroligodendrocyte Space glycoprotein Morn5 MORN5 MORN repeat Other othercontaining 5 Pigz NCBP2 nuclear cap binding Nucleus other proteinsubunit 2, 20 kDa Nptxr NPTXR neuronal pentraxin Plasma transmembranereceptor Membrane receptor Ntm NTM neurotrimin Plasma other MembraneNutf2 NUTF2 nuclear transport Nucleus transporter factor 2 Ocln OCLNoccludin Plasma enzyme Membrane Olr1 OLR1 oxidized low density Plasmatransmembrane lipoprotein (lectin- Membrane receptor like) receptor 1Pabpc4 PABPC4 poly(A) binding Cytoplasm translation protein, cytoplasmicregulator 4 (inducible form) Pde11a PDE11A phosphodiesterase Cytoplasmenzyme dyphylline, 11A nitroglycerin, aminophylline, dipyridamole,tolbutamide, tadalafil, theophylline, pentoxifylline Pdyn PDYNprodynorphin Extracellular transporter Space Per3 PER3 period circadianNucleus other clock 3 Pllp PLLP plasmolipin Plasma transporter MembranePpp1r14c PPP1R14C protein phosphatase Cytoplasm other 1, regulatory(inhibitor) subunit 14C Pramel6 Pramel6 preferentially Other otherexpressed antigen in melanoma like 6 Ptpn18 PTPN18 protein tyrosineNucleus phosphatase phosphatase, non- receptor type 18 (brain-derived)Pycr1 PYCR1 pyrroline-5- Cytoplasm enzyme carboxylate reductase 1 Rab26RAB26 RAB26, member Plasma enzyme RAS oncogene Membrane family Ramp2RAMP2 receptor (G protein- Plasma transporter pramlintide coupled)activity Membrane modifying protein 2 Rbm24 RBM24 RNA binding motifOther other protein 24 Rhag RHAG Rh-associated Plasma peptidaseglycoprotein Membrane Rpl3 RPL3 ribosomal protein Cytoplasm otherhomoharringtonine L3 Sall3 SALL3 sal-like 3 Nucleus other (Drosophila)Satb1 SATB1 SATB homeobox 1 Nucleus transcription regulator Scg2 SCG2secretogranin II Extracellular cytokine Space Slc15a1 SLC15A1 solutecarrier family Plasma transporter 15 (oligopeptide Membranetransporter), member 1 Slc1a1 SLC1A1 solute carrier family Plasmatransporter riluzole 1 Membrane (neuronal/epithelial high affinityglutamate transporter, system Xag), member 1 Slc24a5 Slc24a5 solutecarrier family Other other 24 (sodium/potassium/ calcium exchanger),member 5 Slc37a2 SLC37A2 solute carrier family Other transporter 37(glucose-6- phosphate transporter), member 2 40424 SNTB1 syntrophin,beta 1 Plasma other (dystrophin- Membrane associated protein A1, 59 kDa,basic component 1) St6galnac3 ST6GALNAC3 ST6 (alpha-N- Cytoplasm enzymeacetyl-neuraminyl- 2,3-beta-galactosyl- 1,3)-N- acetylgalactosaminidealpha-2,6- sialyltransferase 3 Tex12 TEX12 testis expressed 12 Nucleusother Tex15 TEX15 testis expressed 15 Extracellular other Space Tfap2aTFAP2A transcription factor Nucleus transcription AP-2 alpha regulator(activating enhancer binding protein 2 alpha) Tmc1 TMC1 transmembranePlasma other channel-like 1 Membrane Tmem130 TMEM130 transmembrane Otherother protein 130 Tmem30b TMEM30B transmembrane Other other protein 30BTomm20 TOMM20 translocase of outer Cytoplasm transporter mitochondrialmembrane 20 homolog (yeast) Tox3 TOX3 TOX high mobility Other othergroup box family member 3 Ttc25 TTC25 tetratricopeptide Cytoplasm otherrepeat domain 25 Tymp TYMP thymidine Extracellular growth phosphorylaseSpace factor Ubb Ubb ubiquitin B Cytoplasm other Vamp7 VAMP7vesicle-associated Cytoplasm transporter membrane protein 7 Wfdc12Wfdc12 WAP four-disulfide Extracellular other core domain 12 SpaceWfdc15a Wfdc15a WAP four-disulfide Other other core domain 15A Wfdc6aWfdc6a WAP four-disulfide Other other core domain 6A

TABLE 14 A subset of genes from Table 13 which are expressed at levels20-fold higher in rat ES cells than the corresponding genes in mouse EScells. ID Entrez Gene Name Ajap1 Adherens Junctions Associate Protein 1Cldn5 Claudin 5 Arhgef9 Cdc42 guanine nucleotide exchange facter 9 Camk4Calcium/calmodulin- dependent protein kinase IV Efna1 ephrin-A1 Epha4EPH receptor A4 Gjb5 gap junction protein beta 5 Igfbpl1 Insulin-likegrowth factor binding protein-like 1 Il1f8 Interleukin 36 beta Il28raInterleukin 28 receptor, alpha Lefty1 left-right determination factor 1Lifr Leukemia inhibitory factor receptor alpha Lpar2 Lysophosphatidicacid receptor 2 Ntm Neuronal pentraxin receptor Ptpn18 Protein tyrosinephosphatase non-receptor type 18 Cdx2 Caudal type homeobox 2 Fank1Fibronectin type III and ankyrin repeat domains 1 Foxe1 Forkhead box E1(thyroid transcription factor 2) Hey2 Hairy/enhancer-of-split relatedwith YRPW motif 2 Lef1 Lymphoid enhancer- binding factor 1 Sall3Sal-like 3 (Drosophila) Satb1 SATB homeobox 1

An additional molecular signature employing the pluripotencymarkers/genes for the rat ES cells has also been developed. Table 15provides a gene list and their expression ranks from the RNA profilingdata. mRNA was isolated from rat ES cells and the expression level ofvarious pluripotency markers were compared relative to each other. The“pluripotency genes” which are listed are genes that other groups haveused (mostly in mouse, but also in rat) as markers of ES cells. Mesodermendoderm and neural are similarly defined. By “rank” is refers to theexpression in our experiment: the higher the rank (1 is highest) thehigher the expression. For example, Oct4's rank of 13 means that, of allthe genes assayed, it was expressed higher than all but 12 genes.Background in this experiment was any expression value below 30; 6107genes had expression values of 30 or higher.

TABLE 15 Rat ES cell molecular signature employing the pluripotency,mesodermal, endodermal, neural and trophectoderm markers/genes.Pluripotency Mesodermal Endodermal Neural Trophectoderm PluripotencyRank Mesodermal Rank Endodermal Rank Neural Rank Trophectoderm Rankc-Myc 8248 Brachyury 7542 Gata6 11195 Nestin 7761 Cdx2 739 Dnmt3L 127Flk1 Not tested Sox17 11418 Pax6 13570 Dppa2 Not tested Nodal 3050 Hhex14571 Sox2 681 Dppa5 Not tested Bmp4 3072 Nodal 3050 Ecat1 9714 Bmpr26382 Ext1 6091 Eras 2541 Sox7 10284 Err-beta 1368 Fbxo15 1369 Fgf4 3440Fthl17 Not tested Gdf3 2771 Rank > 6107 = bkg expression Klf4 836 Lef11313 LIF receptor 724 Lin28 828 Nanog 774 Oct4 13 Rexo1 6119 Sox15 4524Sox2 681 SSEA1 Not tested SSEA4 Not tested Stella Not tested Tcl1 Nottested Utf1 1501

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference. Unless otherwise apparentfrom the context of any embodiment, aspect, step or feature of theinvention can be used in combination with any other. Reference to arange includes any integers within the range, any subrange within therange. Reference to multiple ranges includes composites of such ranges.

That which is claimed:
 1. A method of making a genetically modified ratembryonic stem (ES) cell clone comprising a targeted geneticmodification and capable of generating a genetically modified ratcomprising the targeted genetic modification and transmitting thetargeted genetic modification through the germline, comprising: (a)culturing a population of rat ES cells under conditions comprising alayer of feeder cells that are not modified to express leukemiainhibitory factor (LIF) and a medium comprising about 50 U/mL to about150 U/mL LIF, N2 supplement, B27 supplement, and a combination ofinhibitors consisting of MEK inhibitor PD0325901 and GSK3 inhibitorCHIR99021; (b) introducing into the rat ES cells a polynucleotide orpolypeptide encoding a nuclease agent that generates a single or doublestrand break at a targeted genomic locus and/or a targeting vectorcomprising upstream and downstream homology arms flanking an insertpolynucleotide, wherein the homology arms correspond to genomic regionswithin the targeted genomic locus; and (c) obtaining the geneticallymodified rat ES cell clone comprising the targeted genetic modification,wherein the obtaining consists of identifying in a single cloning stepthe genetically modified rat ES cell clone comprising the targetedgenetic modification and capable of generating the genetically modifiedrat comprising the targeted genetic modification and transmitting thetargeted genetic modification through the germline.
 2. The method ofclaim 1, wherein the LIF is a mouse LIF or has at least 91% sequenceidentity to SEQ ID NO:
 1. 3. The method of claim 1, wherein theconcentration of LIF in the medium is between about 75 U/mL to about 125U/mL.
 4. The method of claim 3, wherein the concentration of LIF in themedium is between about 90 U/mL to about 110 U/mL.
 5. The method ofclaim 4, wherein the concentration of LIF in the medium is about 100U/mL.
 6. The method of claim 5, wherein the concentration of the MEKinhibitor PD0325901 is about 1 μM, and the concentration of the GSK3inhibitor CHIR99021 is about 3 μM.
 7. The method of claim 1, wherein theconcentration of the MEK inhibitor PD0325901 is 0.8 μM to about 1.2 μM,and the concentration of the GSK3 inhibitor CHIR99021 is about 2.5 μM toabout 3 μM or 3 μM to about 3.5 μM.
 8. The method of claim 7, whereinthe concentration of the MEK inhibitor PD0325901 is about 1 μM, and theconcentration of the GSK3 inhibitor CHIR99021 is about 3 μM.
 9. Themethod of claim 1, wherein the layer of feeder cells comprises amonolayer of mitotically inactivated mouse embryonic fibroblasts (MEFs).10. The method of claim 1, wherein the medium further comprises DMEM/F12basal medium and Neurobasal medium.
 11. The method of claim 10, whereinthe layer of feeder cells comprises mitotically inactivated mouseembryonic fibroblasts, and the medium is a 2i medium comprising DMEM/F12basal medium, Neurobasal medium, N2 supplement, B27 supplement, LIF, anda combination of inhibitors consisting of MEK inhibitor PD0325901 andGSK3 inhibitor CHIR99021.
 12. The method of claim 11, wherein theconcentration of the LIF is 100 U/mL, the concentration of the MEKinhibitor PD0325901 is about 1 μM, and the concentration of the GSK3inhibitor CHIR99021 is about 3 μM.
 13. The method of claim 1, wherein:(I) the rat ES cells have a normal karyotype; and/or (II) the rat EScells form spherical, free-floating colonies in culture.
 14. The methodof claim 1, wherein the rat ES cells are derived from an ACI rat or aDark Agouti (DA) rat.
 15. The method of claim 1, wherein the rat EScells are male (XY) rat ES cells.
 16. The method of claim 1, wherein therat ES cells are female (XX) rat ES cells.
 17. The method of claim 1,wherein the rat ES cells have one or more of the followingcharacteristics: (a) the rat ES cells express at least one pluripotencymarker selected from Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4,Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, and Utf1; (b) therat ES cells do not express one or more pluripotency markers selectedfrom c-Myc, Ecat1, and Rexo1; (c) the rat ES cells do not express one ormore mesodermal markers selected from Brachyury and Bmpr2; (d) the ratES cells do not express one or more endodermal markers selected fromGata6, Sox17, and Sox7; (e) the rat ES cells do not express one or moreneural markers selected from Nestin and Pax6; (f) the rat ES cellsexpress one or more pluripotency markers selected from Oct-4, Sox2, andalkaline phosphatase; and (g) the rat ES cells express one or more ratES-cell-specific genes selected from Adherens Junctions AssociatedProtein 1 (Ajap1), Claudin 5 (Cldn5), Cdc42 guanine nucleotide exchangefactor 9 (Arhgef9), Calcium/calmodulin-dependent protein kinase IV(Camk4), ephrin-A1 (Efna1), EPH receptor A4 (Epha4), gap junctionprotein beta 5 (Gjb5), Insulin-like growth factor binding protein-like 1(Igfbpl1), Interleukin 36 beta Interleukin 28 receptor, alpha (Il28ra),left-right determination factor 1 (Lefty1), Leukemia inhibitory factorreceptor alpha (Lifr), Lysophosphatidic acid receptor 2 (Lpar2),Neuronal pentraxin receptor (Ntm), Protein tyrosine phosphatasenon-receptor type 18 (Ptpn18), Caudal type homeobox 2 (Cdx2),Fibronectin type III and ankyrin repeat domains 1 (Fank1), Forkhead boxE1 (thyroid transcription factor 2) (Foxe1), Hairy/enhancer-of-splitrelated with YRPW motif 2 (Hey2), Forkhead box E1 (thyroid transcriptionfactor 2) (Foxe1), Hairy/enhancer-of-split related with YRPW motif 2(Hey2), Lymphoid enhancer-binding factor 1 (Lef1), Sal-like 3(Drosophila) (Sall3), SATB homeobox 1 (Satb1), and miR-632.
 18. Themethod of claim 1, wherein the step (b) comprises one or more rounds ofelectroporation.
 19. The method of claim 1, wherein the targeted geneticmodification comprises an insertion, a deletion, a knockout, a knockin,a point mutation, or a combination thereof.
 20. The method of claim 19,wherein the targeted genetic modification comprises an insertion of aheterologous polynucleotide into the genome of the rat ES cells.
 21. Themethod of claim 1, wherein the method comprises modifying the populationof rat ES cells to comprise two or more targeted modifications, whereinthe modified rat ES cells can transmit the two or more targeted geneticmodifications through the germline.
 22. The method of claim 1, whereinthe targeted genetic modification is generated via a homologousrecombination event.
 23. The method of claim 22, wherein the targetedgenetic modification is generated by employing the targeting vector. 24.The method of claim 1, wherein the targeted genetic modification isgenerated using the nuclease agent.
 25. The method of claim 24, whereinthe nuclease agent is a transcription activator-like effector nuclease(TALEN), a zinc-finger nuclease (ZFN), a meganuclease, or a CRISPR/Cassystem.
 26. The method of claim 1, wherein: (i) step (b) comprisesintroducing into the population of rat ES cells a heterologouspolynucleotide comprising a selection marker, wherein the selectionmarker is operably linked to a promoter active in the rat ES cells, andwherein the selection marker is present in the targeting vector; and(ii) step (c) comprises culturing in vitro the rat ES cells produced bystep (b) in alternating first and second culture media, wherein thefirst culture medium comprises an effective amount of a selection agentfor a first time period and the second culture medium does not comprisethe selection agent, wherein the in vitro culture conditions aresufficient to maintain pluripotency, thereby selecting modified rat EScells having the heterologous polynucleotide stably integrated into thegenome.
 27. The method of claim 26, wherein the first and second culturemedia are alternated every 24 hours.
 28. The method of claim 26, whereinthe selection marker imparts resistance to an antibiotic.
 29. The methodof claim 26, wherein the selection marker comprises one or more ofneomycin phosphotransferase (neo^(r)), hygromycin B phosphotransferase(hyg^(r)), puromycin-N-acetyltransferase (puro^(r)), blasticidin Sdeaminase (bsr^(r)), xanthine/guanine phosphoribosyl transferase (gpt),and herpes simplex virus thymidine kinase (HSV-tk).
 30. The method ofclaim 26, wherein the selection marker has one or more of the followingcharacteristics: (a) the selection marker comprises a non-attenuatedselection marker gene; and (b) the selection marker has an increasedactivity compared to a wild type selection marker.
 31. The method ofclaim 26, wherein the modified rat ES cells comprise multiple copies ofthe selection marker stably incorporated into the genome.
 32. The methodof claim 1, wherein the concentration of LIF in the medium is between 50U/mL to 150 U/mL.
 33. The method of claim 32, wherein the concentrationof LIF in the medium is between 75 U/mL to 125 U/mL.
 34. The method ofclaim 33, wherein the concentration of LIF in the medium is between 90U/mL to 110 U/mL.
 35. The method of claim 34, wherein the concentrationof LIF in the medium is 100 U/mL.
 36. A method of making a geneticallymodified rat, comprising: (a) making a genetically modified ratembryonic stem (ES) cell clone according to the method of claim 1; (b)introducing the rat ES cell clone from step (a) into a rat host embryo;(c) gestating the rat host embryo comprising the rat ES cell clone in asurrogate mother, wherein the surrogate mother produces an F0 progenygenetically modified rat comprising the targeted genetic modification;and (d) breeding the F0 progeny genetically modified rat with anotherrat to produce an F1 progeny genetically modified rat comprising thetargeted genetic modification, wherein the targeted genetic modificationis transmitted through the germline.
 37. The method of claim 36, whereinstep (d) comprises breeding a male F0 rat identified in step (c) with awild type female rat to produce an F1 progeny that is heterozygous forthe targeted genetic modification.
 38. The method of claim 37, furthercomprising: (e) breeding a male rat of the F1 progeny with a female ratof the F1 progeny to obtain an F2 progeny that is homozygous for thetargeted genetic modification.
 39. The method of claim 37, wherein atleast 3%, at least 10%, or at least 60% of the F1 progeny are derivedfrom the rat ES cell clone in step (a).